Tagged oligonucleotides and their use in nucleic acid amplification methods

ABSTRACT

The present invention provides nucleic acid amplification systems and methods that desirably reduce or eliminate false positive amplification signals resulting from contaminating biological material, e.g., nucleic acid, that may be present in one or more reagents used in an amplification reaction and/or that may be present in the environment in which an amplification reaction is performed. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in amplification reactions, and the environment in which an amplification reaction is performed, are free of bacterial or other nucleic acid contamination that may yield false positive results.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. No. 8,580,510, filedSep. 12, 2012, which is a continuation of U.S. Pat. No. 8,278,052, filedSep. 13, 2011, which is a continuation of U.S. Pat. No. 8,034,570, filedSep. 28, 2010, which is a divisional application of U.S. Pat. No.7,833,716, filed Jun. 6, 2007, which claims priority to U.S. ProvisionalApplication No. 60/811,581, filed Jun. 6, 2006, and U.S. ProvisionalApplication No. 60/871,442, filed Dec. 21, 2006, the contents of eachbeing incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to methods, compositions, reaction mixtures andkits for the selective amplification of multiple copies of a specificnucleic acid sequence or “target sequence” which may be present eitheralone or as a component of a homogeneous or heterogeneous mixture ofnucleic acids. The mixture of nucleic acids may be that found in asample taken for diagnostic testing, screening of blood products,sterility testing, microbiological detection in food, water, beverage,industrial or environmental samples, research studies, preparingreagents or materials for other processes such as cloning, or for otherpurposes. The selective amplification of specific nucleic acidsequences, as described herein, is of particular value in any of avariety of detection assays for increasing the accuracy and reliabilityof such assays while at the same time reducing the preparation,purification and/or sterilization requirements for reagents used in theassays and for the environment in which the assays are performed.

DESCRIPTION OF THE RELATED ART

The detection and/or quantitation of specific nucleic acid sequences isan important technique for identifying and classifying microorganisms,diagnosing infectious diseases, measuring response to various types oftreatment, and the like. Such procedures are also useful in detectingand quantitating microorganisms in foodstuffs, water, beverages,industrial and environmental samples, seed stocks, and other types ofmaterial where the presence of specific microorganisms may need to bemonitored.

Numerous amplification-based methods for the detection and quantitationof target nucleic acids are well known and established in the art. Thepolymerase chain reaction, commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of thetarget sequence (e.g., Mullis et al., “Process for Amplifying, Detectingand/or Cloning Nucleic Acid Sequences,” U.S. Pat. No. 4,683,195; Mullis,“Process for Amplifying Nucleic Acid Sequences,” U.S. Pat. No.4,683,202; Mullis et al., “Process for Amplifying, Detecting and/orCloning Nucleic Acid Sequences,” U.S. Pat. No. 4,800,159; Gelfand etal., “Reaction Mixtures for the Detection of Target Nucleic Acids,” U.S.Pat. No. 5,804,375; Mullis et al. (1987) Meth. Enzymol. 155, 335-350;and Murakawa et al. (1988) DNA 7, 287-295).

In a variation called RT-PCR, reverse transcriptase (RT) is used to makea complementary DNA (cDNA) from RNA, and the cDNA is then amplified byPCR to produce multiple copies of DNA (Gelfand et al., “ReverseTranscription with Thermostable DNA Polymerases—High Temperature ReverseTranscription,” U.S. Pat. Nos. 5,322,770 and 5,310,652).

Another well known amplification method is strand displacementamplification, commonly referred to as SDA, which uses cycles ofannealing pairs of primer sequences to opposite strands of a targetsequence, primer extension in the presence of a dNTP to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product (e.g., Walker, G. et al. (1992),Proc. Natl. Acad. Sci. USA 89, 392-396; Walker et al., “Nucleic AcidTarget Generation,” U.S. Pat. No. 5,270,184; Walker, “StrandDisplacement Amplification,” U.S. Pat. No. 5,455,166; and Walker et al.(1992) Nucleic Acids Research 20, 1691-1696). Thermophilic SDA (tSDA)uses thermophilic endonucleases and polymerases at higher temperaturesin essentially the same method (European Pat. No. 0 684 315).

Other amplification methods include rolling circle amplification (RCA)(e.g., Lizardi, “Rolling Circle Replication Reporter Systems,” U.S. Pat.No. 5,854,033); helicase dependent amplification (HDA) (e.g., Kong etal., “Helicase Dependent Amplification Nucleic Acids,” U.S. Pat. Appln.Pub. No. US 2004-0058378 A1); and loop-mediated isothermal amplification(LAMP) (e.g., Notomi et al., “Process for Synthesizing Nucleic Acid,”U.S. Pat. No. 6,410,278).

Transcription-based amplification methods commonly used in the artinclude nucleic acid sequence based amplification, also referred to asNASBA (e.g., Malek et al., U.S. Pat. No. 5,130,238); methods which relyon the use of an RNA replicase to amplify the probe molecule itself,commonly referred to as Qβ replicase (e.g., Lizardi, P. et al. (1988)BioTechnol. 6, 1197-1202); transcription-based amplification methods(e.g., Kwoh, D. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173-1177)and self-sustained sequence replication (e.g., Guatelli, J. et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874-1878; Landgren (1993) Trendsin Genetics 9, 199-202; and HELEN H. LEE et al., NUCLEIC ACIDAMPLIFICATION TECHNOLOGIES (1997)).

Another transcription-based amplification method istranscription-mediated amplification, commonly referred to as TMA, whichsynthesizes multiple copies of a target nucleic acid sequenceautocatalytically under conditions of substantially constanttemperature, ionic strength, and pH, in which multiple RNA copies of thetarget sequence autocatalytically generate additional copies (e.g.,Kacian et al., “Nucleic Acid Sequence Amplification Methods,” U.S. Pat.No. 5,480,784; and Kacian et al., U.S. Pat. No. 5,399,491). TMA is arobust and highly sensitive amplification system with demonstratedefficacy, which overcomes many of the problems associated with PCR-basedamplification systems. In particular, temperature cycling is notrequired.

Amplification assays are particularly well suited for the detection ofmicroorganisms in the context of clinical laboratory testing, bioprocessmonitoring, or any other setting in which the detection ofmicroorganisms in a particular sample type is desired, by offering highsensitivity and rapid time-to-result relative to conventionalmicrobiological techniques. In addition, amplification methods can beused in the detection of the vast number of microorganisms that aredifficult or impossible to culture on synthetic media. Nevertheless,there are certain limitations associated with first-generationamplification assays that have limited their acceptance in certainsettings, such as clinical microbiological laboratories. One inherentproblem associated with the high sensitivity of nucleic acidamplification systems is that contaminating nucleic acid introduced intothe amplification system (e.g., from one or more reagents used duringamplification, from the technician performing the assay, from theenvironment in which the amplification is performed, etc.) can result infalse positive results. For example, even extremely small amounts ofnucleic acid contamination present in reagents and/or enzymes used in anamplification reaction, or in the environment in which the amplificationreaction is performed, can give rise to a positive amplification signaldespite the fact that the sequence of interest is not present in thenucleic acid sample being tested. This requires that significant effortbe expended in sample preparation, purification, sterilization, etc., ofthe reagents used in amplification reactions to avoid or minimize falsepositive results.

Accordingly, there remains a need in the art for a robust nucleic acidamplification system that can selectively amplify one or more targetnucleic acid sequences of interest while reducing or eliminating falsepositive results that can arise as a result of contaminating biologicalmaterial, such as contaminating nucleic acid. There also remains a needfor amplification systems that have reduced reagent purification and/orsterility requirements. As described further herein, the presentinvention meets these needs and offers other related advantages.

SUMMARY OF THE INVENTION

The present invention is directed generally to nucleic acidamplification methods and reaction mixtures that desirably reduce oreliminate false positive amplification signals resulting fromcontaminating biological material, e.g., nucleic acid, that may bepresent in one or more reagents, components or materials that are usedin an amplification reaction or that are present in the environment inwhich an amplification reaction is performed. The invention furtheroffers the advantage of requiring less stringent purification and/orsterility efforts than conventionally needed in order to ensure thatenzymes and other reagents and components used in amplificationreactions are free of bacterial and other nucleic acid contaminationthat may yield false positive results. Such components or materialsinclude, but are not limited to, water, buffers, salts, solid supports(e.g., magnetically charged particles or beads), and receptacles (e.g.,glassware or plasticware). Accordingly, the methods and reactionmixtures of the invention are useful in detecting and/or quantitatingmicroorganisms in clinical samples, foodstuffs, water, industrial andenvironmental samples, seed stocks, and other types of material wherethe presence of microorganisms may need to be detected and/or monitored.The methods and reaction mixtures of the invention have particularadvantages for the testing raw materials used in the production ofproducts for the biotech, pharma, cosmetics and beverage industries, forrelease testing of final products, and for sterility screening to testfor a class of organisms or total viable organisms in a material ofinterest (bacterial, fungal or both). In the clinical setting, themethods and reaction mixtures of the invention would be particularlyuseful for sepsis testing, especially septicemia, which is caused bypathogenic organisms and/or their toxins in the bloodstream.

According to one embodiment of the present invention, there are providedmethods for the selective amplification of at least one target nucleicacid sequence, such as a DNA sequence or an RNA sequence, where themethod comprises the steps of: (a) treating a target nucleic acidsequence in a nucleic acid sample, e.g., where the target nucleic acidis immobilized on a solid support, with a heterologous tag sequence toproduce a tagged target nucleic acid sequence; (b) reducing in saidsample the effective concentration of heterologous tag sequences whichhave not formed part of said tagged target nucleic acid sequence and arein a form capable of producing a tagged target nucleic acid sequencewith said target nucleic acid sequence; and (c) subjecting said taggedtarget nucleic acid sequence to reagents and conditions sufficient fordetectable amplification of the target nucleic acid sequence, where thesubjecting step exposes the nucleic acid sample to a known contaminatingsource of the target nucleic acid sequence after step (b), and wheredetectable amplification of the target nucleic acid sequence issubstantially limited to amplification of target nucleic acid sequencecontributed by the tagged target nucleic acid sequence of step (a) andnot by the target nucleic acid sequence contributed by the knowncontaminating source.

The methods of the invention are particularly useful where one or morereagents or components used are produced with a material known to be acontaminating source of a target nucleic acid sequence being amplified.In one example, one or more reagents used in the methods, such asnucleic acid polymerases, are produced using a microorganism containingthe target nucleic acid sequence. In another example, components used inthe methods, such as reaction vessels, pipette tips and solid supportsfor binding the tagged target nucleic acid sequences, may be a knowncontaminating source of the target nucleic acid sequence. In addition,the methods are useful where the environmental conditions in whichamplification is performed include a known contaminating source of atarget nucleic acid sequence, such as the ambient air, operator oranalytical instrumentation.

In a more particular aspect of this embodiment, the tagged targetnucleic acid sequence is immobilized on a solid support during step (b).

In another particular aspect, step (b) comprises diluting or removingheterologous tag sequences which have not formed part of the taggedtarget nucleic acid sequence from the nucleic acid sample. In analternative aspect, step (b) comprises inactivating heterologous tagsequences which have not formed part of said tagged target nucleic acidsequence to produce an inactivated heterologous tag sequence. In arelated aspect, the method further comprises removing the inactivatedheterologous tag sequence from said nucleic acid sample during step (b).The heterologous tag sequence may be inactivated by blocking its abilityto complex with the target nucleic acid sequence, using an enzyme todigest a component or cleave a site of a complexed portion of theheterologous tag sequence, chemically altering the heterologous tagsequence, or altering by other means the ability of the heterologous tagsequence to complex with the target nucleic acid sequence in anamplification reaction mixture.

In another aspect, the heterologous tag sequence is contained in atagged oligonucleotide, where the tagged oligonucleotide comprises firstand second regions, the first region comprising a target hybridizingsequence which hybridizes to a 3′-end of the target nucleic acidsequence and the second region comprising a tag sequence situated 5′ tothe target hybridizing sequence, and where the tag sequence does notstably hybridize to a target nucleic acid containing the target nucleicacid sequence.

In yet another aspect, the heterologous tag sequence has an active formduring step (a) which permits the heterologous tag sequence to producethe tagged target nucleic acid sequence, and where the heterologous tagsequence which has not produced the tagged target nucleic acid sequenceis converted to an inactive form in step (b) which blocks theheterologous tag sequence from producing a tagged target nucleic acidsequence during step (c).

The target hybridizing sequence, in certain aspects, is a universaloligonucleotide, such as a universal bacterial or fungaloligonucleotide.

Step (c) comprises producing amplification products in a nucleic acidamplification reaction using first and second oligonucleotides, thefirst oligonucleotide comprising a sequence which hybridizes to a 3′-endof the complement of the target nucleic acid sequence and the secondoligonucleotide comprising a sequence which hybridizes to a complementof the tag sequence but which does not stably hybridize to the targetnucleic acid sequence, wherein each of the amplification productscomprises a base sequence which is substantially identical orcomplementary to the base sequence of the target nucleic acid sequenceand further comprises a base sequence which is substantially identicalor complementary to all or a portion of the tag sequence.

Various amplification methods are suitable for use in the presentinvention. For example, in one aspect, the amplification reaction is aPCR reaction. In another aspect, the target nucleic acid sequence isamplified by a transcription-based amplification reaction, preferably aTMA reaction, performed under isothermal conditions.

The target nucleic acid sequence amplified according to the methods canbe any target nucleic acid sequence of interest, but will generally be anucleic acid sequence obtained from a microorganism. Further, the methodcan be selective for the amplification of a target nucleic acid sequencecontained in the nucleic acid of a single strain or species ofmicroorganisms or in multiple species of microorganisms. Alternatively,the method can be selective for the amplification of multiple targetnucleic acid sequences contained in the nucleic acid of multiple speciesof microorganisms, where, for example, the target hybridizing sequenceof a tagged oligonucleotide hybridizes to a target region present ineach of the multiple target nucleic acid sequences in step (a).

For example, in a particular aspect, the method is selective for theamplification of a target nucleic acid sequence contained in each of aplurality of target nucleic acids, and wherein the heterologous tagsequence produces a tagged target nucleic acid sequence with the targetnucleic acid sequence of each of the plurality of target nucleic acidspresent in the nucleic acid sample in step (a). In a more particularaspect, the target nucleic acid sequence contained in each of theplurality of target nucleic acids is the same nucleic acid sequence.

In another particular aspect, the method is selective for theamplification of multiple bacterial or fungal target nucleic acidsequences, e.g., wherein the multiple bacterial or fungal target nucleicacid sequences are ribosomal nucleic acid sequences.

In another particular aspect, the method is selective for theamplification of target nucleic acid sequences obtained from members ofa group of bacterial species including Staphylococci spp. (e.g.,Staphylococcus aureus, Staphylococcus epidermis and Staphylococcushaemolyticus), Steptococci spp. (e.g., Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis,Viridans streptococci and beta-hemolytic streptococci), Enterococcusspp. (e.g., Enterococcus faecium and Enterococcus faecalis), Escherichiaspp. (e.g., Escherichia coli), Klebsiella spp. (e.g., Klebsiellapneumoniae and Klebsiella oxytoca), Pseudomonas spp. (e.g., Pseudomonasaeruginosa), Enterobacter spp. (e.g., Enterobacter cloacae andEnterobacter aerogenes), Proteus spp. (e.g., Proteus mirabilis),Bacterioides spp., Clostridium spp., Serratia spp. (e.g., Serratiamarcescens), Acinetobacter spp. (e.g., Acinetobacter baumannii) andStenotrophomonas spp. (e.g., Stenotrophomonas maltophilia). At least aportion of these microorganisms would be appropriate for detection in asepsis test.

In another aspect, the method is selective for the amplification oftarget nucleic acid sequences obtained from members of a group of fungalspecies including Candida spp. (e.g., Candida albicans, Candidatropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae,Candida krusei, Candida zeylanoides, Candida guilliermondi, Candidapseudotropicalis and Candida famata), Histoplama capsulatum,Cryptococcus spp. (e.g., Cryptococcus neoformans, Cryptococcus albidusand Cryptococcus laurentii) Coccidioides spp. (e.g., Coccidioidesimmitis), Trichosporon spp. (e.g., Trichosporon cutaneum), Malasseziaspp. (e.g., Malassezia furfur), Rhodotorula spp., Nocardia spp. (e.g.,Nocardia asteroides), Fusarium spp. and Asperigillus spp. (e.g.,Asperigillus fumigatus). At least a portion of these microorganismswould be appropriate for detection in a sepsis test.

In yet another aspect, at least a portion of a nucleic acid sample usedin the methods is obtained from a clinical, water, industrial,environmental, seed, beverage or food source.

The methods are particularly well suited, in certain aspects, for use insterility testing or diagnostic testing for sepsis.

According to another embodiment of the invention, there is provided amethod for the selective amplification of at least one target nucleicacid sequence from a nucleic acid sample, the method comprising thesteps of: (a) treating a nucleic acid sample comprising a target nucleicacid sequence with a tagged oligonucleotide comprising first and secondregions, the first region comprising a target hybridizing sequence whichhybridizes to a 3′-end of the target nucleic acid sequence and thesecond region comprising a tag sequence situated 5′ to the targethybridizing sequence, where the second region does not stably hybridizeto a target nucleic acid containing the target nucleic acid sequence;(b) reducing in said nucleic acid sample the effective concentration ofunhybridized tagged oligonucleotide having an active form in which atarget hybridizing sequence of said unhybridized tagged oligonucleotideis available for hybridization to said target nucleic acid sequence; and(c) producing amplification products in a nucleic acid amplificationreaction using first and second oligonucleotides, where the firstoligonucleotide comprises a hybridizing sequence which hybridizes to a3′-end of the complement of the target nucleic acid sequence and thesecond oligonucleotide comprises a hybridizing sequence which hybridizesto the complement of the tag sequence, where the second oligonucleotidedoes stably hybridize to the target nucleic acid, and where each of theamplification products comprises a base sequence which is substantiallyidentical or complementary to the base sequence of the target nucleicacid sequence and further comprises a base sequence which issubstantially identical or complementary to all or a portion of the tagsequence.

In one aspect of the above methods, at least one target nucleic acidsequence is immobilized on a solid support during step (b). In anotheraspect, step (b) does not include the use of an enzyme having a nucleaseactivity.

The effective concentration of unhybridized tagged oligonucleotide in anactive form prior to amplification is preferably reduced by diluting thenucleic acid sample or by inactivating and/or removing the unhybridizedtagged oligonucleotide. In one aspect, step (b) comprises inactivatingunhybridized tagged oligonucleotide so that the unhybridized taggedoligonucleotide does not stably hybridize to the target nucleic acidsequence during step (c). In one example of inactivation, a taggedoligonucleotide has an active form during step (a) which permits thetarget hybridizing sequence to hybridize to the target nucleic acidsequence, and where unhybridized tagged oligonucleotide is converted toan inactive form in step (b) which blocks or prevents the taggedoligonucleotide from hybridizing to the target nucleic acid sequenceduring step (c). The tagged oligonucleotide may be inactivated byblocking the target hybridizing sequence from hybridizing to the targetnucleic acid sequence, using an enzyme to digest a component or cleave asite of a duplex formed between the target hybridizing sequence and thetarget nucleic acid sequence, chemically altering the target hybridizingsequence, or altering by other means the ability of the taggedoligonucleotide to hybridize to the target nucleic acid sequence in anamplification reaction mixture.

In a related embodiment, the conditions of steps (b) and (c) are lessstringent than the conditions of step (a). In another relatedembodiment, the temperature of the nucleic acid sample is loweredbetween steps (a) and (b).

In another example where step (b) comprises inactivating unhybridizedtagged oligonucleotide, unhybridized tagged oligonucleotide from step(a) is converted from a single-stranded form to a duplexed form in step(b). The duplexed form may be a hairpin tag molecule comprising a tagclosing sequence joined to a 5′-end of the tagged oligonucleotide, wherethe tag closing sequence hybridizes to the target hybridizing sequenceunder the conditions of step (b), thereby blocking hybridization ofunhybridized tagged oligonucleotide from step (a) to the target nucleicacid sequence in steps (b) and (c). In another aspect, the tag closingsequence is joined to the tagged oligonucleotide by a non-nucleotidelinker. For example, a 5′-end of the tag closing sequence may be joinedto a 5′-end of the tagged oligonucleotide.

The tagged oligonucleotide can also further comprise a third regioncontaining a promoter for an RNA polymerase, the third region beingsituated 5′ to the second region.

In another aspect, the tag closing sequence is modified to prevent theinitiation of DNA synthesis therefrom.

According to another aspect, a 3′-terminal base of the targethybridizing sequence is hybridized to a 5′-terminal base of the tagclosing sequence. In another aspect, a 3′-end of the tag closingsequence is joined to a 5′-end of the tagged oligonucleotide.

In still another aspect, the target hybridizing sequence is hybridizedto a tag closing oligonucleotide in step (b), the tagged oligonucleotideand the tag closing oligonucleotide being distinct molecules. The tagclosing oligonucleotide may be modified, if desired, to prevent theinitiation of DNA synthesis therefrom.

Further, in certain aspects, a 3′-terminal base of the targethybridizing sequence is hybridized to a 5′-terminal base of the tagclosing oligonucleotide.

In certain other aspects, the tagged oligonucleotide and the tag closingoligonucleotide are both present in the nucleic acid sample during step(a), and where the target hybridizing sequence favors hybridization tothe target nucleic acid sequence over the tag closing oligonucleotide instep (a).

As noted above, the methods of the invention can employ any of a varietyof amplification techniques. In certain instances it may be preferredthat an isothermal amplification reaction is used, such as atranscription-based amplification reaction, preferably TMA or real-timeTMA.

In a particular aspect, the first oligonucleotide comprises a promoterfor an RNA polymerase which is situated 5′ to the hybridizing sequence.In another aspect, the second oligonucleotide comprises a promoter foran RNA polymerase which is situated 5′ to the hybridizing sequence, andwhere the tagged oligonucleotide further comprises a promoter for an RNApolymerase which is situated 5′ to the second region.

The target nucleic acid sequence amplified according to the methods canbe any target nucleic acid sequence of interest, but will generally be anucleic acid sequence obtained from a microorganism. Further, the methodcan be selective for the amplification of a target nucleic acid sequencecontained in the nucleic acid of a single strain or species ofmicroorganisms or in multiple species of microorganisms. Alternatively,the method can be selective for the amplification of multiple targetnucleic acid sequences contained in the nucleic acid of multiple speciesof microorganisms, where, for example, the target hybridizing sequenceof a tagged oligonucleotide hybridizes to a target region present ineach of the multiple target nucleic acid sequences in step (a).

In another aspect, the method is selective for the amplification of atarget nucleic acid sequence contained in each of a plurality of targetnucleic acids, and wherein the target hybridizing sequence hybridizes toa 3′-end of the target nucleic acid sequence of each of the plurality oftarget nucleic acids present in the nucleic acid sample in step (a). Inanother aspect, the target nucleic acid sequence contained in each ofsaid plurality of target nucleic acids is the same nucleic acidsequence.

In a particular embodiment, the method is selective for theamplification of multiple bacterial or fungal target nucleic acidsequences, e.g., wherein the multiple bacterial or fungal target nucleicacid sequences are ribosomal nucleic acid sequences.

In a more particular embodiment, the method is selective for theamplification of target nucleic acid sequences obtained from members ofa group of bacterial species including Staphylococci spp. (e.g.,Staphylococcus aureus, Staphylococcus epidermis and Staphylococcushaemolyticus), Steptococci spp. (e.g., Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis,Viridans streptococci and beta-hemolytic streptococci), Enterococcusspp. (e.g., Enterococcus faecium and Enterococcus faecalis), Escherichiaspp. (e.g., Escherichia coli), Klebsiella spp. (e.g., Klebsiellapneumoniae and Klebsiella oxytoca), Pseudomonas spp. (e.g., Pseudomonasaeruginosa), Enterobacter spp. (e.g., Enterobacter cloacae andEnterobacter aerogenes), Proteus spp. (e.g., Proteus mirabilis),Bacterioides spp., Clostridium spp., Serratia spp. (e.g., Serratiamarcescens), Acinetobacter spp. (e.g., Acinetobacter baumannii) andStenotrophomonas spp. (e.g., Stenotrophomonas maltophilia). At least aportion of these microorganisms would be appropriate for detection in asepsis test.

In another particular embodiment, the method is selective for theamplification of target nucleic acid sequences obtained from members ofa group of fungal species including Candida spp. (e.g., Candidaalbicans, Candida tropicalis, Candida glabrata, Candida parapsilosis,Candida lusitaniae, Candida krusei, Candida zeylanoides, Candidaguilliermondi, Candida pseudotropicalis and Candida famata), Histoplamacapsulatum, Cryptococcus spp. (e.g., Cryptococcus neoformans,Cryptococcus albidus and Cryptococcus laurentii) Coccidioides spp.(e.g., Coccidioides immitis), Trichosporon spp. (e.g., Trichosporoncutaneum), Malassezia spp. (e.g., Malassezia furfur), Rhodotorula spp.,Nocardia spp. (e.g., Nocardia asteroides), Fusarium spp. andAsperigillus spp. (e.g., Asperigillus fumigatus). At least a portion ofthese microorganisms would be appropriate for detection in a sepsistest.

In certain aspects, the target hybridizing sequence hybridizes to a3′-end of each of multiple target nucleic acid sequences present in thenucleic acid sample in step (a). Further, the first oligonucleotidehybridizes to a 3′-end of the complement of each of the multiple targetnucleic acid sequences present in the nucleic acid sample in step (c).

The method can also comprise a plurality of first oligonucleotides, eachof the plurality of first oligonucleotides hybridizing to a 3′-end ofthe complement of at least one but less than all of the multiple targetnucleic acid sequences present in the nucleic acid sample in step (c).

The tagged oligonucleotide, in a particular embodiment of the invention,is a universal bacterial oligonucleotide or a universal fungaloligonucleotide. Such tagged oligonucleotides are particularly wellsuited to methods for sterility testing, such as methods for analyzingbioprocess materials or which are diagnostic for sepsis.

In a more particular aspect, the target hybridizing sequence hybridizesto a 3′-end of the target nucleic acid of each of the plurality oftarget nucleic acids present in the nucleic acid sample in step (a), theplurality of target nucleic acids belonging to a class of microorganismsselected from the group consisting of Eubacteria, Gram-positivebacteria, Gram-negative bacteria and fungi. In another aspect, each ofplurality of target nucleic acids is a ribosomal nucleic acid.

In another aspect of the invention, the multiple target nucleic acidsequences include members belonging to a class of bacterialmicroorganisms selected from the group consisting of Staphylococci spp.(e.g., Staphylococcus aureus, Staphylococcus epidermis andStaphylococcus haemolyticus), Steptococci spp. (e.g., Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus mitis, Viridans streptococci and beta-hemolyticstreptococci), Enterococcus spp. (e.g., Enterococcus faecium andEnterococcus faecalis), Escherichia spp. (e.g., Escherichia coli),Klebsiella spp. (e.g., Klebsiella pneumoniae and Klebsiella oxytoca),Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Enterobacter spp.(e.g., Enterobacter cloacae and Enterobacter aerogenes), Proteus spp.(e.g., Proteus mirabilis), Bacterioides spp., Clostridium spp., Serratiaspp. (e.g., Serratia marcescens), Acinetobacter spp. (e.g.,Acinetobacter baumannii) and Stenotrophomonas spp. (e.g.,Stenotrophomonas maltophilia). At least a portion of thesemicroorganisms would be appropriate for detection in a sepsis test.

In a further aspect of the invention, the multiple target nucleic acidsequences include members belonging to a class of fungal microorganismsselected from the group consisting of Candida spp. (e.g., Candidaalbicans, Candida tropicalis, Candida glabrata, Candida parapsilosis,Candida lusitaniae, Candida krusei, Candida zeylanoides, Candidaguilliermondi, Candida pseudotropicalis and Candida famata), Histoplamacapsulatum, Cryptococcus spp. (e.g., Cryptococcus neoformans,Cryptococcus albidus and Cryptococcus laurentii) Coccidioides spp.(e.g., Coccidioides immitis), Trichosporon spp. (e.g., Trichosporoncutaneum), Malassezia spp. (e.g., Malassezia furfur), Rhodotorula spp.,Nocardia spp. (e.g., Nocardia asteroides), Fusarium spp. andAsperigillus spp. (e.g., Asperigillus fumigatus). At least a portion ofthese microorganisms would be appropriate for detection in a sepsistest.

The nucleic acid sample is often exposed to a known contaminating sourceof the target nucleic acid sequence after step (b), and, accordingly,the described methods provide that the production of amplificationproducts is substantially limited to amplification of target nucleicacid sequence contributed by the nucleic acid sample and not by thecontaminating source of the target nucleic acid sequence. For example,one or more reagents or components used in the amplification reaction isa known contaminating source of the target nucleic acid sequence.Alternatively, or in addition, one or more reagents are produced with amaterial known to be a contaminating source of the target nucleic acidsequence, such as nucleic acid polymerases produced using microorganismsknown to contain the target nucleic acid sequence. Further, theenvironmental conditions in which the method is performed may include aknown contaminating source of the target nucleic acid sequence. In aparticular aspect, at least a portion of said nucleic acid is obtainedfrom a clinical, water, industrial, environmental, seed, beverage orfood source.

According to another embodiment of the present invention, the targetnucleic acid sequence is an RNA target sequence, and step (c) comprises:extending the tagged oligonucleotide hybridized to the target nucleicacid sequence in a primer extension reaction with a DNA polymerase toproduce a first primer extension product comprising a regioncomplementary to the target nucleic acid sequence; separating the firstprimer extension product from the target nucleic acid using an enzymewhich selectively degrades that portion of the target nucleic acidhybridized to the first primer extension product; treating the firstprimer extension product with the first oligonucleotide, the firstoligonucleotide being a promoter oligonucleotide comprising first andsecond regions, the first region comprising a hybridizing sequence whichhybridizes to a region of the first primer extension product that iscomplementary to a 5′-end of the target nucleic acid sequence to form apromoter oligonucleotide:first primer extension product hybrid, and thesecond region comprising a promoter for an RNA polymerase which issituated 5′ to the first region; transcribing from the promoteroligonucleotide:first primer extension product hybrid multiple copies ofa first RNA product complementary to at least a portion of the firstprimer extension product using an RNA polymerase which recognizes thepromoter and initiates transcription therefrom, where the base sequenceof the first RNA product is substantially identical to the base sequenceof the target nucleic acid sequence and the complement of the tagsequence; treating the first RNA product with the secondoligonucleotide, the second oligonucleotide being a primingoligonucleotide which hybridizes to the complement of the tag sequenceto form a priming oligonucleotide:first RNA product hybrid such that aprimer extension reaction can be initiated from the primingoligonucleotide; extending the priming oligonucleotide in a primerextension reaction with a DNA polymerase to produce a second primerextension product complementary to the first RNA product, the secondprimer extension product having a 3′-end which is complementary to a5′-end of the first RNA product; separating the second primer extensionproduct from the first RNA product using an enzyme which selectivelydegrades said first RNA product; treating the second primer extensionproduct with the promoter oligonucleotide to form a promoteroligonucleotide:second primer extension product hybrid; extending a3′-end of the second primer extension product in the promoteroligonucleotide:second primer extension product hybrid to add a sequencecomplementary to the second region of the promoter oligonucleotide; andtranscribing from the promoter oligonucleotide:second primer extensionproduct hybrid multiple copies of a second RNA product complementary tothe second primer extension product using the RNA polymerase, whereinthe base sequence of the second RNA product is substantially identicalto the base sequence of the target nucleic acid sequence and thecomplement of the tag sequence.

In another aspect of this embodiment of the invention, step (a) furthercomprises treating the nucleic acid sample with a binding molecule whichbinds to the target nucleic acid adjacent to or near a 5′-end of thetarget nucleic acid sequence, and where the first primer extensionproduct has a 3′-end which is determined by the binding molecule andwhich is complementary to the 5′-end of the target nucleic acidsequence.

In another aspect, step (c) of the above embodiment further comprisesextending a 3′-end of the first primer extension product in the promoteroligonucleotide:first primer extension product hybrid to add a sequencecomplementary to the promoter. In yet another aspect, the promoteroligonucleotide is modified to prevent the initiation of DNA synthesistherefrom.

The promoter oligonucleotide hybridized to the first primer extensionproduct is extended with a DNA polymerase to produce a primer extensionproduct complementary to the first primer extension product; and thepromoter oligonucleotide hybridized to said second primer extensionproduct is extended with a DNA polymerase to produce a primer extensionproduct complementary to the second primer extension product.

The separating steps of the described methods may be performed with aribonuclease activity provided by the DNA polymerase. Alternatively, theseparating steps are performed with a ribonuclease activity provided byan enzyme other than said DNA polymerase.

According to another embodiment of the present invention, the targetnucleic acid sequence is an RNA target sequence, and step (c) comprises:extending the tagged oligonucleotide hybridized to the target nucleicacid sequence in a primer extension reaction with a DNA polymerase toproduce a first primer extension product comprising a regioncomplementary to the target nucleic acid sequence, where the taggedoligonucleotide further comprises a third region situated 5′ to thesecond region, the third region comprising a promoter for an RNApolymerase; separating the first primer extension product from thetarget nucleic acid using an enzyme which selectively degrades thatportion of the target nucleic acid hybridized to the first primerextension product; treating the first primer extension product with thefirst oligonucleotide, the first oligonucleotide being a primingoligonucleotide which hybridizes to a region of the first primerextension product that is complementary to a 5′-end of the targetnucleic acid sequence to form a priming oligonucleotide:first primerextension product hybrid such that a primer extension reaction can beinitiated from the priming oligonucleotide; extending the primingoligonucleotide in a primer extension reaction with a DNA polymerase toproduce a second primer extension product complementary to the firstprimer extension product; and using the second primer extension productas a template to transcribe multiple copies of a first RNA productcomplementary to at least a portion of the second primer extensionproduct using an RNA polymerase which recognizes the promoter andinitiates transcription therefrom, where the base sequence of the firstRNA product is substantially identical to the base sequence of the tagsequence and the complement of the target nucleic acid sequence.

In another aspect of this embodiment, step (c) further comprises:treating the first RNA product with the priming oligonucleotide to forma priming oligonucleotide:first RNA product hybrid such that a primerextension reaction can be initiated from the priming oligonucleotide;extending the priming oligonucleotide in a primer extension reactionwith a DNA polymerase to produce a third primer extension productcomplementary to the first RNA product, the third primer extensionproduct having a 3′-end which is complementary to a 5′-end of the firstRNA product; separating the third primer extension product from thefirst RNA product using an enzyme which selectively degrades the firstRNA product; treating the third primer extension product with the secondoligonucleotide, the second oligonucleotide being a promoteroligonucleotide comprising first and second regions, the first regioncomprising a hybridizing sequence which hybridizes to the complement ofthe tag sequence to form a promoter oligonucleotide:third primerextension product hybrid such that a primer extension reaction can beinitiated from the promoter oligonucleotide, and the second regioncomprising a promoter for an RNA polymerase which is situated 5′ to thefirst region; extending the promoter oligonucleotide in a primerextension reaction with the DNA polymerase to produce a fourth primerextension product complementary to the third primer extension product;extending the third primer extension product to add a sequencecomplementary to the promoter; transcribing from the promoteroligonucleotide:third primer extension product hybrid multiple copies ofa second RNA product complementary to the third primer extension productusing an RNA polymerase which recognizes the promoter and initiatestranscription therefrom, where the base sequence of the second RNAproduct is substantially identical to the base sequence of the tagsequence and the complement of the target nucleic acid sequence.

In another aspect of this embodiment, the separating steps are performedwith a ribonuclease activity provided by the DNA polymerase.Alternatively, the separating steps are performed with a ribonucleaseactivity provided by an enzyme other than the DNA polymerase.

According to another embodiment of the present invention, the targetnucleic acid sequence is a DNA target sequence, and step (c) comprises:extending the tagged oligonucleotide hybridized to the target nucleicacid sequence in a primer extension reaction with a DNA polymerase toproduce a first primer extension product comprising a regioncomplementary to the target nucleic acid sequence; treating the firstprimer extension product with the first oligonucleotide, the firstoligonucleotide being a promoter oligonucleotide comprising first andsecond regions, the first region comprising a hybridizing sequence whichhybridizes to a region of the first primer extension product that iscomplementary to a 5′-end of the target nucleic acid sequence to form apromoter oligonucleotide:first primer extension product hybrid, and thesecond region being a promoter for an RNA polymerase which is situated5′ to the first region; transcribing from the promoteroligonucleotide:first primer extension product hybrid multiple copies ofa first RNA product complementary to at least a portion of the firstprimer extension product using an RNA polymerase which recognizes thepromoter and initiates transcription therefrom, where the base sequenceof the first RNA product is substantially identical to the base sequenceof the target nucleic acid sequence and the complement of the tagsequence; treating the first RNA product with the secondoligonucleotide, the second oligonucleotide being a primingoligonucleotide which hybridizes to the complement of the tag sequenceto form a priming oligonucleotide:first RNA product hybrid such that aprimer extension reaction can be initiated from the primingoligonucleotide; extending the priming oligonucleotide in a primerextension reaction with a DNA polymerase to give a second primerextension product comprising the complement of the first RNA product,the second primer extension product having a 3′-end which iscomplementary to a 5′-end of the first RNA product; separating thesecond primer extension product from the first RNA product using anenzyme which selectively degrades the first RNA product; treating thesecond primer extension product with the promoter oligonucleotide toform a promoter oligonucleotide:second primer extension product hybrid;extending a 3′-end of the second primer extension product in thepromoter oligonucleotide:second primer extension product hybrid to add asequence complementary to the promoter; and transcribing from thepromoter oligonucleotide:second primer extension product hybrid multiplecopies of a second RNA product complementary to the second primerextension product using the RNA polymerase, where the base sequence ofthe second RNA product is substantially identical to the base sequenceof the target nucleic acid sequence and the complement of the tagsequence.

In one aspect of this embodiment, the promoter oligonucleotide ismodified to prevent the initiation of DNA synthesis therefrom.

In another aspect, step (a) further comprises: treating the nucleic acidsample with a displacer oligonucleotide which hybridizes to the targetnucleic acid upstream from the tagged oligonucleotide such that a primerextension reaction can be initiated from the displacer oligonucleotide;and extending the displacer oligonucleotide in a primer extensionreaction with a DNA polymerase to produce a third primer extensionproduct that displaces said first primer extension product from thetarget nucleic acid.

In yet another embodiment, step (a) further comprises treating thenucleic acid sample with a binding molecule which binds to the targetnucleic acid adjacent to or near a 5′-end of the target nucleic acidsequence, where the first primer extension product has a 3′-end which isdetermined by said binding molecule and which is complementary to the5′-end of the target nucleic acid sequence.

In a more particular aspect, step (c) further comprises extending a3′-end of the first primer extension product in the promoteroligonucleotide:first primer extension product hybrid to add a sequencecomplementary to the promoter sequence.

In another particular aspect, step (c) further comprises: extending thepromoter oligonucleotide hybridized to the first primer extensionproduct with a DNA polymerase to produce a primer extension productcomplementary to the first primer extension product; and extending thepromoter oligonucleotide hybridized to the second primer extensionproduct with a DNA polymerase to produce a primer extension productcomplementary to the second primer extension product.

The separating steps, in one embodiment, are performed by a ribonucleaseactivity provided by said DNA polymerase. Alternatively, the separatingsteps are performed by a ribonuclease activity provided by an enzymeother than said DNA polymerase.

Another embodiment of the present invention provides a kit for use inthe selective amplification of at least one target nucleic acid sequencefrom a nucleic acid sample, the kit comprising: a tagged oligonucleotidecomprising a first region comprising a target hybridizing sequence whichhybridizes to a 3′-end of a target nucleic acid sequence under a firstset of conditions so that the first region can be extended in atemplate-dependent manner in the presence of a DNA polymerase, and asecond region comprising a tag sequence situated 5′ to the first region,where the second region does not stably hybridize to a target nucleicacid containing the target nucleic acid sequence under the first set ofconditions; a tag closing sequence which hybridizes to the targethybridizing sequence under a second set of conditions, thereby blockinghybridization of the tagged oligonucleotide to the target nucleic acidsequence, where the tag closing sequence does not stably hybridize tothe target hybridizing sequence under the first set of conditions; and afirst priming oligonucleotide which hybridizes to the complement of thetag sequence under the second set of conditions so that the firstpriming oligonucleotide can be extended in a template-dependent mannerin the presence of a DNA polymerase.

In a more particular aspect of this embodiment, the taggedoligonucleotide further comprises a third region containing a promoterfor an RNA polymerase, the third region being situated 5′ to the secondregion.

In another aspect, the 3′-terminal base of the target hybridizingsequence hybridizes to a 5′-terminal base of the tag closing sequencewhen the target hybridizing sequence is not hybridized to the targetnucleic acid sequence under the second set of conditions.

In yet another aspect, the 5′-end of the tag closing sequence includes amoiety for stabilizing a duplex formed between the tag closing sequenceand the target hybridizing sequence when the target hybridizing sequenceis not hybridized to the target nucleic acid sequence under the secondset of conditions.

In another aspect, the tagged oligonucleotide and the tag closingsequence constitute distinct molecules, the tag closing sequence being atag closing oligonucleotide. Alternatively, the tagged oligonucleotideand the tag closing sequence are contained in the same molecule.

The tag closing sequence may be joined to the tagged oligonucleotide bya non-nucleotide linker, for example a non-nucleotide linker comprisingat least one of abasic nucleotides and polyethylene glycol.

In another aspect, a 3′-end of the tag closing sequence is joined to a5′-end of the tagged oligonucleotide. Alternatively, a 5′-end of the tagclosing sequence is joined to a 5′-end of the tagged oligonucleotide.

In yet another aspect, the tag closing sequence hybridizes to the targethybridizing sequence to form an antiparallel duplex when the targethybridizing sequence is not hybridized to the target nucleic acidsequence under the second set of conditions.

In another aspect, the tag closing sequence is modified to prevent theinitiation of DNA synthesis therefrom, for example by including ablocking moiety situated at its 3′-terminus.

In another aspect, the tag closing sequence hybridizes to the targethybridizing sequence to form a parallel duplex when the targethybridizing sequence is not hybridized to the target nucleic acidsequence under the second set of conditions.

In yet another aspect, the duplex comprises a 3′-terminal base of thetarget hybridizing sequence hybridized to a 3′-terminal base of the tagclosing sequence.

The tag closing sequence, in this aspect, may be modified to prevent theinitiation of DNA synthesis therefrom, for example by including ablocking moiety situated at its 3′-terminus.

In another aspect of this embodiment, the first priming oligonucleotidedoes stably hybridize to the target nucleic acid and, thereby,participates in detectable amplification of the target nucleic acidsequence under the second set of conditions.

In another aspect, the kit further comprises a second primingoligonucleotide which hybridizes to the complement of a 5′-end of thetarget nucleic acid sequence under the second set of conditions so thatthe second priming oligonucleotide can be extended in atemplate-dependent manner in the presence of a DNA polymerase.

In yet another aspect, a kit of the invention further comprises apromoter oligonucleotide comprising first and second regions, the firstregion comprising a hybridizing sequence which hybridizes to thecomplement of a 5′-end of the target nucleic acid sequence under thesecond set of conditions, and the second region comprising a promoterfor an RNA polymerase which is situated 5′ to the first region.

The promoter oligonucleotide, in this aspect, may be modified to preventthe initiation of DNA synthesis therefrom, for example by including ablocking moiety situated at its 3′-terminus.

In yet another aspect, the promoter oligonucleotide can be extended in atemplate-dependent manner in the presence of a DNA polymerase when thehybridizing sequence is hybridized to the complement of the 5′-end ofthe target nucleic acid sequence under the second set of conditions.

The kits of the invention, in certain aspects, may also further compriseone or more reagents or components selected from any one or more of aDNA polymerase (such as a reverse transcriptase), an RNA polymerase,nucleoside triphosphates, a solid support for binding a complexcomprising the target nucleic acid and the tagged oligonucleotide. Inanother aspect, the tagged oligonucleotide is free in solution.

In another aspect, the kit does not include a restriction enzyme capableof cleaving a duplex formed between the tag closing sequence and thetarget hybridizing sequence under the second set of conditions.

In yet another aspect, the target hybridizing sequence hybridizes to a3′-end of multiple target nucleic acid sequences under the first set ofconditions.

In another embodiment, the tagged oligonucleotide is a universalbacterial or a universal fungal oligonucleotide. For example, in oneaspect, the target hybridizing sequence hybridizes to target region at a3′-end of one or more target nucleic acid sequences, the target regionbeing present in a plurality of microorganisms belonging to a class ofmicroorganisms selected from the group consisting of Eubacteria,Gram-positive bacteria, Gram-negative bacteria and fungi under the firstset of conditions. In another embodiment, said one or more targetnucleic acid sequences are ribosomal nucleic acid sequences.

In a more particular aspect, the microorganisms belong to a class ofbacterial microorganisms selected from the group consistingStaphylococci spp. (e.g., Staphylococcus aureus, Staphylococcusepidermis and Staphylococcus haemolyticus), Steptococci spp. (e.g.,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus mitis, Viridans streptococci andbeta-hemolytic streptococci), Enterococcus spp. (e.g., Enterococcusfaecium and Enterococcus faecalis), Escherichia spp. (e.g., Escherichiacoli), Klebsiella spp. (e.g., Klebsiella pneumoniae and Klebsiellaoxytoca), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Enterobacterspp. (e.g., Enterobacter cloacae and Enterobacter aerogenes), Proteusspp. (e.g., Proteus mirabilis), Bacterioides spp., Clostridium spp.,Serratia spp. (e.g., Serratia marcescens), Acinetobacter spp. (e.g.,Acinetobacter baumannii) and Stenotrophomonas spp. (e.g.,Stenotrophomonas maltophilia). At least a portion of thesemicroorganisms would be appropriate for detection in a sepsis test.

In another particular aspect, the microorganisms belong to a fungalgroup of microorganisms selected from the group consisting of Candidaspp. (e.g., Candida albicans, Candida tropicalis, Candida glabrata,Candida parapsilosis, Candida lusitaniae, Candida krusei, Candidazeylanoides, Candida guilliermondi, Candida pseudotropicalis and Candidafamata), Histoplama capsulatum, Cryptococcus spp. (e.g., Cryptococcusneoformans, Cryptococcus albidus and Cryptococcus laurentii)Coccidioides spp. (e.g., Coccidioides immitis), Trichosporon spp. (e.g.,Trichosporon cutaneum), Malassezia spp. (e.g., Malassezia furfur),Rhodotorula spp., Nocardia spp. (e.g., Nocardia asteroides), Fusariumspp. and Asperigillus spp. (e.g., Asperigillus fumigatus). At least aportion of these microorganisms would be appropriate for detection in asepsis test.

According to another embodiment of the invention, there is providedreaction mixture for amplifying a target nucleic acid sequence, thereaction mixture comprising: a tagged oligonucleotide comprising firstand second regions, the first region comprising a target hybridizingsequence hybridized to a 3′-end of a target nucleic acid sequence andthe second region comprising a tag sequence situated 5′ to the targethybridizing sequence; a first oligonucleotide comprising a hybridizingsequence which hybridizes to a 3′-end of the complement of the targetnucleic acid sequence; and a second oligonucleotide comprising ahybridizing sequence which hybridizes to the complement of the tagsequence, where unhybridized tagged oligonucleotide in the reactionmixture has an inactive form which blocks or prevents the unhybridizedtagged oligonucleotide from hybridizing to the target nucleic acidsequence.

In a more particular aspect according to this embodiment, the inactiveform of the tagged oligonucleotide comprises a tag closing sequencehybridized to the target hybridizing sequence.

In another aspect, the tagged oligonucleotide and said tag closingsequence are distinct molecules, the tag closing sequence being a tagclosing oligonucleotide.

In another aspect, the tagged oligonucleotide and the tag closingsequence are contained in the same molecule.

In yet another aspect, the tagged oligonucleotide is not attached to asolid support.

Certain other embodiments of the invention relate to the use of themethods described herein as a means for monitoring bioprocess samples,streams, and the like. In one embodiment, for example, there is provideda method for monitoring a bioprocess for the presence of contaminatingnucleic acid comprising the steps of (a) treating a first bioprocesssample with a tagged oligonucleotide, wherein said taggedoligonucleotide comprises first and second regions, the first regioncomprising a target hybridizing sequence capable of hybridizing to atarget nucleic acid sequence of an organism of interest and the secondregion comprising a tag sequence, situated 5′ to said target hybridizingsequence, which does not stably hybridize to the target nucleic acidsequence; under conditions wherein the tagged oligonucleotide stablyhybridizes to the target nucleic acid sequence present in said firstsample; (b) removing or inactivating unhybridized tagged oligonucleotidefrom the first bioprocess sample; and (c) exposing a second bioprocesssample, the second bioprocess sample comprising the first bioprocesssample and further comprising additional bioprocess samples, toamplification reagents and conditions sufficient for amplification ofthe target nucleic acid sequence using: (i) a first oligonucleotidewhich hybridizes to a complement of the tag sequence and (ii) a secondoligonucleotide sequence which hybridizes to a complement of the targetnucleic acid sequence, where detectable amplification resulting from thefirst and second oligonucleotides is contributed by the target nucleicacid sequence of an organism of interest in the first bioprocess sampleand not by the target nucleic acid sequence contributed by theadditional bioprocess samples.

In another embodiment, the present invention provides a method formonitoring a bioprocess for the presence of contaminating nucleic acidcomprising the steps of (a) treating a first bioprocess sample with afirst tagged oligonucleotide, where the first tagged oligonucleotidecomprises first and second regions, the first region comprising a targethybridizing sequence capable of hybridizing to a target nucleic acidsequence of an organism of interest and the second region comprising afirst tag sequence, situated 5′ to the target hybridizing sequence,which does not stably hybridize to the target nucleic acid sequence;under conditions where the first tagged oligonucleotide stablyhybridizes to the target nucleic acid sequence present in said firstsample; (b) treating a second bioprocess sample with a second taggedoligonucleotide, where the second tagged oligonucleotide comprises firstand second regions, the first region comprising a target hybridizingsequence capable of hybridizing to the target nucleic acid sequence ofthe organism of interest and the second region comprising a second tagsequence, situated 5′ to the target hybridizing sequence and differentfrom the first tag sequence, which does not stably hybridize to thetarget nucleic acid sequence; under conditions where the second taggedoligonucleotide stably hybridizes to the target nucleic acid sequencepresent in the second sample; and (c) performing a nucleic acidamplification reaction on a third bioprocess sample, the thirdbioprocess sample comprising the first and the second bioprocesssamples, using: (i) a first oligonucleotide which hybridizes to acomplement of the first tag sequence; (ii) a second oligonucleotidesequence which hybridizes to a complement of the second tag sequence;and (iii) a third oligonucleotide which hybridizes to a complement ofthe target nucleic acid sequence; where the detection of amplificationproduct resulting from the first and second oligonucleotides isindicative of the presence of the target nucleic acid sequence of theorganism of interest in the first bioprocess sample, and where detectionof amplification product resulting from the first and thirdoligonucleotides is indicative of the presence of the target nucleicacid sequence of the organism of interest in the second bioprocesssample.

In a further embodiment of the invention, a pre-amplification reactionmixture is provided for the selective amplification of one or moretarget nucleic acid sequences, where the reaction mixture comprises: atagged oligonucleotide comprising first and second regions, said firstregion comprising a target hybridizing sequence hybridized to a targetregion contained at a 3′-end of one or more target nucleic acidsequences present in the reaction mixture and the second regioncomprises a tag sequence situated 5′ to the target hybridizing sequence;a first oligonucleotide comprising a hybridizing sequence whichhybridizes to a 3′-end of the complement of one or more of the targetnucleic acid sequences; and a second oligonucleotide comprising ahybridizing sequence which hybridizes to the complement of the tagsequence, where the second oligonucleotide preferably does not stablyhybridize to a target nucleic acid containing the target nucleic acidsuch that it can be enzymatically extended in the presence of a nucleicacid polymerase added to the reaction mixture to produce a primerextension product complementary to one or more of the target nucleicacid sequences, where the reaction mixture is substantially free of anactive form of the tagged oligonucleotide which is not hybridized to thetarget region contained in the one or more target nucleic acid sequencespresent in the reaction mixture, where the active form of the taggedoligonucleotide has an available target hybridizing sequence forhybridization to the target region present in a non-target nucleic acidadded to the reaction mixture, and where the reaction mixture does notinclude a nucleic acid polymerase capable of extending any of theoligonucleotides in a template-dependent manner. The “non-target”nucleic is from a source outside of the reaction mixture and may containa sequence identical to that of the target nucleic acid sequence. Thesource of the non-target nucleic acid may be environmental or it may bea component or reagent added to the reaction mixture, such a nucleicacid polymerase. The tagged oligonucleotide can be a tagged primingoligonucleotide or a tagged promoter oligonucleotide having a promoterrecognized by an RNA polymerase situated 5′ to the second region.

In one aspect of this embodiment, the tagged priming oligonucleotidedoes not include a promoter for RNA polymerase, while the firstoligonucleotide includes an RNA promoter situated 5′ to the hybridizingsequence of the first oligonucleotide. In a preferred aspect, the firstoligonucleotide further includes a blocking moiety situated at its3′-terminus. In another aspect that is useful for amplifying an E. colitarget sequence, the target hybridizing sequence of the taggedoligonucleotide consists of SEQ ID NO:19, its RNA equivalent or acomplement thereof, and the first hybridizing sequence of the firstoligonucleotide consists of SEQ ID NO:20, its RNA equivalent or acomplement thereof.

In another aspect of this embodiment, the tagged oligonucleotide and thefirst oligonucleotide may each include a promoter for an RNA polymerasesituated 5′ to the tag sequence and the hybridizing sequence,respectively. In this aspect, the first oligonucleotide can include ablocking moiety situated at its 3′-terminus.

In yet another aspect of this embodiment, the tagged oligonucleotideincludes a tag closing sequence joined to its 3′-end, thereby forming aunitary molecule referred to as a “tag molecule.” Depending on thenature of the amplification reaction, the tag molecule may or may notinclude a promoter for an RNA polymerase situated 5′ to the tagsequence. In one embodiment, tag molecules that have not hybridized tothe target region of at least one target nucleic acid sequence remain“free” in the reaction mixture (i.e., the tag molecules do not formhybrid duplexes other than through self-hybridization). Self-hybridizedtag molecules are referred to as “hairpin tag molecules,” which is aninactive form of the tag molecule that prevents it from hybridizing toany complementary nucleic acids that are subsequently added to thereaction mixture, such as through a contaminated enzyme preparation orreagent containing non-target nucleic acids. In still another aspect ofthis embodiment, substantially all of the tag molecules in the reactionmixture are in a hybridized state (hybridized either to the targetregion of a target nucleic acid sequence or to themselves in the form ofhairpin tag molecules). At least a portion of the tag molecules whichhave not hybridized to the target region of a target nucleic acidsequence (i.e., hairpin tag molecules) are removed from the reactionmixture by, for example, subjecting the reaction mixture to a targetcapture and washing procedure.

In a still further aspect of this embodiment, there are substantially notagged oligonucleotides that exist in an unhybridized state when thereaction mixture is exposed to an enzyme preparation for amplifying theone or more target nucleic acid sequences. Thus, in this aspect, thereaction mixture is substantially depleted of unhybridized taggedoligonucleotides specific for the one or more target nucleic acidsequences provided by the sample of interest. This may be accomplishedwith, for example, a target capture and washing procedure that separateshybridized tagged oligonucleotides from unhybridized taggedoligonucleotides, and then selectively removes the unhybridized taggedoligonucleotides from the reaction mixture.

In yet another aspect of this embodiment, the tagged oligonucleotidedoes not include either a tag closing sequence or a tag closingoligonucleotide. Accordingly, in this aspect the tagged oligonucleotidecannot be characterized as being a “tag molecule.”

In still another aspect of this embodiment, a probe is included fordetecting an amplification product synthesized in an in vitro reactionthat involves enzymatic extension of the tagged oligonucleotide and thesecond oligonucleotide. The amplification product includes copies of oneor more of the target nucleic acid sequences and/or their complements.

In yet another embodiment, a reaction mixture is provided for theselective amplification of one or more target nucleic acid sequences,where the reaction mixture comprises: a tagged oligonucleotidecomprising first and second regions, where the first region comprises atarget hybridizing sequence hybridized to a 3′-end of a target nucleicacid sequence and the second region comprises a tag sequence situated 5′to the target hybridizing sequence; a first oligonucleotide comprising ahybridizing sequence which hybridizes to a 3′-end of the complement ofthe target nucleic acid sequence; and a second oligonucleotidecomprising a hybridizing sequence which hybridizes to the complement ofthe tag sequence, where the second oligonucleotide preferably does notstably hybridize to a target nucleic acid containing the target nucleicacid such that it can be enzymatically extended in the presence of anucleic acid polymerase added to or present in the reaction mixture toproduce a primer extension product complementary to one or more of thetarget nucleic acid sequences, and where substantially all unhybridizedtagged oligonucleotide in the reaction mixture has an inactive formwhich blocks or prevents said unhybridized tagged oligonucleotide fromhybridizing to the target nucleic acid sequence.

The inactive form of the tagged oligonucleotide can comprise a tagclosing sequence hybridized to the target hybridizing sequence. The tagclosing sequence can be a distinct molecule when not hybridized to thetarget hybridizing sequence or it can be contained in a moleculeincluding the tagged oligonucleotide, in which case the tag closingsequence is preferably joined to the 5′-end of the taggedoligonucleotide by a non-nucleotide linker (i.e., the constituents ofthe linker cannot be copied by a nucleic acid polymerase). The taggedoligonucleotide may or may not be joined to a solid support and ispreferably not directly attached to solid support (e.g., particles orbeads). If joined to a solid support, either directly or indirectly, thetagged oligonucleotide may further function as a capture probe forbinding and immobilizing a target nucleic acid sequence.

The tagged oligonucleotides of the above reaction mixture embodimentsmay possess the characterizing features of any of the various taggedoligonucleotides embodiments described infra. And, unless specificallyexcluded, the reaction mixtures may further include the reagents andcomponents needed to conduct an amplification reaction.

These and other features and advantages of the present invention willbecome apparent upon reference to the following detailed description,the attached drawings and the claims. All references disclosed hereinare hereby incorporated by reference in their entirety as if each wasincorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the steps of a transcription-based amplificationreaction initiated with a tagged priming oligonucleotide that hybridizesto a 3′-end of an RNA target sequence. A first extension product formedwith the tagged priming oligonucleotide has a 3′-end which is determinedby a terminating oligonucleotide hybridized adjacent to or near the5′-end of the RNA target sequence. A blocked promoter oligonucleotidehybridizes to a 3′-end of the first extension product and is used togenerate RNA transcripts that are cycled into the amplificationreaction.

FIG. 2 illustrates the use of a hairpin tag molecule in theamplification reaction of FIG. 1.

FIGS. 3A and 3B illustrate the steps of a transcription-mediatedamplification reaction initiated with a tagged promoter oligonucleotidethat hybridizes to a 3′-end of an RNA target sequence.

FIG. 4 illustrates the use of a hairpin tag molecule in theamplification reaction of FIGS. 3A and 3B.

FIG. 5 illustrates the steps of a transcription-based amplificationreaction initiated with a tagged priming oligonucleotide that hybridizesto a 3′-end of a single-stranded DNA target sequence. A first extensionproduct formed with the tagged priming oligonucleotide has a 3′-endwhich is determined by a terminating oligonucleotide hybridized adjacentto or near the 5′-end of the DNA target sequence. A displaceroligonucleotide hybridized 5′ to the tagged priming oligonucleotide isextended to form a second extension product which displaces the firstextension product from the DNA target sequence. A blocked promoteroligonucleotide hybridizes to a 3′-end of the first extension productand is used to generate RNA transcripts that are cycled into theamplification reaction.

FIG. 6 illustrates the use of a hairpin tag molecule in theamplification reaction of FIG. 5.

FIG. 7 illustrates the steps polymerase chain reaction that is initiatedwith a tagged priming oligonucleotide that hybridizes to a DNA targetsequence.

FIG. 8 illustrates the use of a hairpin tag molecule in theamplification reaction of FIG. 7.

FIG. 9 illustrates the steps of a reverse transcription polymerase chainreaction initiated with a tagged priming oligonucleotide that hybridizesto an RNA target sequence.

FIG. 10 illustrates the use of a hairpin tag molecule in theamplification reaction of FIG. 9.

FIG. 11 illustrates a discrete, 3′ blocked tag closing oligonucleotidehybridized in an antiparallel fashion to the 3′-end of a tagged primingoligonucleotide, thereby blocking hybridization of the tagged primingoligonucleotide to a target nucleic acid sequence.

FIG. 12 illustrates a discrete, 3′ blocked tag closing oligonucleotidehybridized in an antiparallel fashion to the 3′-end of a tagged promoteroligonucleotide, thereby blocking hybridization of the tagged promoteroligonucleotide to a target nucleic acid sequence.

FIG. 13 illustrates a hairpin tag molecule that includes a 3′ blockedtag closing sequence hybridized in a parallel fashion to the 3′-end of atagged priming oligonucleotide, thereby blocking hybridization of thetagged priming oligonucleotide to a target nucleic acid sequence. A5′-end of the tag closing sequence is joined to the 3′-end of a tagsequence of the tagged priming oligonucleotide by a non-nucleotidelinker.

FIG. 14 illustrates a hairpin tag molecule that includes a 3′ blockedtag closing sequence hybridized in a parallel fashion to the 3′-end of atagged promoter oligonucleotide, thereby blocking hybridization of thetagged promoter oligonucleotide to a target nucleic acid sequence. A5′-end of the tag closing sequence is joined to the 3′-end of a promotersequence of the tagged promoter oligonucleotide by a non-nucleotidelinker.

FIG. 15 illustrates a hairpin tag molecule that includes a 3′ blockedtag closing sequence hybridized in an antiparallel fashion to the 3′-endof a tagged priming oligonucleotide, thereby blocking hybridization ofthe tagged priming oligonucleotide to a target nucleic acid sequence. A5′-end of the tag closing sequence is joined to the 3′-end of a tagsequence of the tagged priming oligonucleotide by a non-nucleotidelinker.

FIG. 16 illustrates a hairpin tag molecule that includes a 3′ blockedtag closing sequence hybridized in an antiparallel fashion to the 3′-endof a tagged promoter oligonucleotide, thereby blocking hybridization ofthe tagged promoter oligonucleotide to a target nucleic acid sequence. A5′-end of the tag closing sequence is joined to the 3′-end of a promotersequence of the tagged promoter oligonucleotide by a non-nucleotidelinker.

FIG. 17 shows the raw curves for HCV amplifications in which no targetwas spiked into the amplification reagent. There was no detectableamplification when the HCV transcript was not spiked into the targetcapture or amplification reagents, while the average TTime for reactionscontaining 1×10⁶ copies of the HCV transcript in the target capturereagent was 6.3 minutes.

FIG. 18 shows the raw curves for HCV amplifications in which target wasspiked into the amplification reagent. There was no detectableamplification when the HCV transcript was spiked into the amplificationreagent, while the average TTime for reactions containing 1×10⁶ copiesof the HCV transcript in the target capture reagent was 6.3 minutes. Thezero samples in target capture did not amplify, even with 1 millioncopies HCV 1a spiked into the amplification reagent.

FIG. 19 shows the raw curves for HCV amplifications in which target andtagged nonT7 primer were spiked into the amplification reagent. TheAvgTTime for 1 million copies HCV 1a target present only in the targetcapture step with tagged nonT7 primer & terminating oligonucleotidespiked into the amplification reagent was 7.2 minutes. The zero samplesin target capture with target, terminating oligonucleotide & taggednonT7 primer spiked into the amplification reagent also produced robustamplification with an AvgTTime=8.6 minutes.

FIG. 20 is graph showing results from time-dependent monitoring ofnucleic acid amplification reactions that included either 0 or 10⁶copies of a synthetic E. coli rRNA template. The thin broken line showsresults for the reaction conducted using 0 copies of template, and theheavy solid line shows results for the reaction conducted using 10⁶copies of template.

FIG. 21 is graph showing results from time-dependent monitoring ofnucleic acid amplification reactions that included either 0, 10³ or 10⁵copies of a synthetic E. coli rRNA template.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, nucleic acid amplificationmethods are provided that desirably reduce or eliminate false positiveamplification signals resulting from contaminating biological materialthat may be present in a reagent or component of an amplificationreaction. The provided methods also allow for less stringentpurification and/or sterility efforts than have been conventionallyneeded in order to ensure that enzymes and other reagents or componentsused in amplification reactions, and the environment in whichamplification reactions are performed, are free of contamination bymicroorganisms or components thereof, such as nucleic acid material,that may yield false positive results.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, recombinantDNA, and chemistry, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

Definitions

The following terms have the following meanings unless expressly statedto the contrary. It is to be noted that the term “a” or “an” entityrefers to one or more of that entity; for example, “a nucleic acid,” isunderstood to represent one or more nucleic acids. As such, the terms“a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

Nucleic Acid

The term “nucleic acid” is intended to encompass a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, viral genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double-stranded or single-stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases which make up a nucleic acid. The term “polynucleotide” may beused herein to denote a nucleic acid chain. Throughout this application,nucleic acids are designated as having a 5′-terminus and a 3′-terminus.Standard nucleic acids, e.g., DNA and RNA, are typically synthesized“3′-to-5′,” i.e., by the addition of nucleotides to the 5′-terminus of agrowing nucleic acid.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The termalso includes analogs of such subunits, such as a methoxy group at the2′ position of the ribose (2′-O-Me). As used herein, methoxyoligonucleotides containing “T” residues have a methoxy group at the 2′position of the ribose moiety, and a uracil at the base position of thenucleotide.

A “non-nucleotide unit” is a unit that does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

Target Nucleic Acid/Target Sequence

A “target nucleic acid” is a nucleic acid present in a nucleic acidsample comprising a “target sequence” to be amplified. Target nucleicacids may be DNA or RNA as described herein, and may be eithersingle-stranded or double-stranded. The target nucleic acid may includeother sequences besides the target sequence which may not be amplified.Typical target nucleic acids include viral genomes, bacterial genomes,fungal genomes, plant genomes, animal genomes, rRNA, tRNA, or mRNA fromviruses, bacteria or eukaryotic cells, mitochondrial DNA, or chromosomalDNA.

Target nucleic acids may be isolated from any number of sources based onthe purpose of the amplification assay being carried out. Sources oftarget nucleic acids include, but are not limited to, clinical specimens(e.g., blood, either whole blood or platelets, urine, saliva, feces,semen, or spinal fluid), environmental samples (e.g., water or soilsamples), food samples, beverages, industrial samples (e.g., productsand process materials, including water), seed stocks, cDNA libraries, ortotal cellular RNA. By “isolated” it is meant that a sample containing atarget nucleic acid is taken from its natural milieu; however, the termdoes not connote any particular degree of purification. If necessary,target nucleic acids of the present invention are made available forinteraction with the various oligonucleotides of the present invention.This may include, for example, cell lysis or cell permeabilization torelease the target nucleic acid from cells, which then may be followedby one or more purification steps, such as a series of isolation andwash steps. See, e.g., Clark et al., “Method for Extracting NucleicAcids from a Wide Range of Organisms,” U.S. Pat. No. 5,786,208; andHogan, “Polynucleotide Matrix-Based Method of IdentifyingMicroorganisms, U.S. Pat. No. 6,821,770. This may be particularlyimportant where the sample source or cellular material released into thesample can interfere with the amplification reaction. Methods to preparetarget nucleic acids from various sources for amplification are wellknown to those of ordinary skill in the art. Target nucleic acids of thepresent invention may be purified to some degree prior to theamplification reactions described herein, but in other cases, the sampleis added to the amplification reaction without any furthermanipulations.

The term “target sequence” refers to the particular nucleotide sequenceof the target nucleic acid which is to be amplified. The “targetsequence” includes the complexing sequences to which oligonucleotides(e.g., tagged oligonucleotides, priming oligonucleotides and/or promoteroligonucleotides) complex during the processes of the present invention.Where the target nucleic acid is originally single-stranded, the term“target sequence” will also refer to the sequence complementary to the“target sequence” as present in the target nucleic acid. Where the“target nucleic acid” is originally double-stranded, the term “targetsequence” refers to both the sense (+) and antisense (−) strands. Inchoosing a target sequence, the skilled artisan will understand that a“unique” sequence should be chosen so as to distinguish betweenunrelated or closely related target nucleic acids. As will be understoodby those of ordinary skill in the art, “unique” sequences are judgedfrom the testing environment. At least the sequences recognized by thetarget hybridizing sequence of a tagged oligonucleotide and theassociated detection probe or probes (as described in more detailelsewhere herein) should be unique in the environment being tested, butneed not be unique within the universe of all possible sequences.Furthermore, even though the target sequence should contain a “unique”sequence for recognition by a tagged oligonucleotide or detection probe,it is not always the case that the priming oligonucleotide and/orpromoter oligonucleotide are recognizing “unique” sequences. In someembodiments, it may be desirable to choose a target sequence which iscommon to a class of organisms, for example, a sequence which is commonto all E. coli strains that might be in a sample. In other situations, avery highly specific target sequence, or a target sequence having atleast a highly specific region recognized by the detection probe, wouldbe chosen so as to distinguish between closely related organisms, forexample, between pathogenic and non-pathogenic E. coli. A targetsequence of the present invention may be of any practical length. Aminimal target sequence includes a region which hybridizes to the targethybridizing sequence of a tagged oligonucleotide, the complement of aregion which hybridizes to a priming oligonucleotide or the hybridizingregion of a promoter oligonucleotide, and a region used for detection,e.g., a region (or complement thereof) which hybridizes to a detectionprobe, as described in more detail elsewhere herein. The region whichhybridizes with the detection probe may overlap with or be containedwithin the region which hybridizes with the priming oligonucleotide (orits complement) or the hybridizing region of the promoteroligonucleotide (or its complement). In addition to the minimalrequirements, the optimal length of a target sequence depends on anumber of considerations, for example, the amount of secondarystructure, or self-hybridizing regions in the sequence. Determining theoptimal length is easily accomplished by those of ordinary skill in theart using routine optimization methods. Typically, target sequences ofthe present invention range from about 100 nucleotides in length to fromabout 150 to about 250 nucleotides in length. The optimal or preferredlength may vary under different conditions, which can easily be testedby one of ordinary skill in the art according to the methods describedherein. The terms “amplicon” refers to a nucleic acid molecule generatedduring an amplification procedure that is substantially complementary oridentical to a sequence contained within the target sequence. The term“amplification product” refers to an amplicon or some other productindicative of an amplification reaction.

Oligonucleotides

As used herein, the term “oligonucleotide” or “oligo” or “oligomer” isintended to encompass a singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods of the present invention, as wellas subsequent detection methods. The oligonucleotide may be DNA and/orRNA and/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent, rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions, e.g., it may function as a primer if it iscapable of hybridizing to a complementary strand and can further beextended in the presence of a nucleic acid polymerase, it may provide apromoter if it contains a sequence recognized by an RNA polymerase andallows for transcription, and it may function to prevent hybridizationor impede primer extension if appropriately situated and/or modified.Specific oligonucleotides of the present invention are described in moredetail below. As used herein, an oligonucleotide can be virtually anylength, limited only by its specific function in the amplificationreaction or in detecting an amplification product of the amplificationreaction.

Oligonucleotides of a defined sequence and chemical structure may beproduced by techniques known to those of ordinary skill in the art, suchas by chemical or biochemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules, e.g., bacterial orviral vectors. As intended by this disclosure, an oligonucleotide doesnot consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide of the present invention. Modifications include basemodifications, sugar modifications or backbone modifications. Basemodifications include, but are not limited to the use of the followingbases in addition to adenine, cytidine, guanosine, thymine and uracil:C-5 propyne, 2-amino adenine, 5-methyl cytidine, inosine, and dP and dKbases. The sugar groups of the nucleoside subunits may be ribose,deoxyribose and analogs thereof, including, for example, ribonucleosideshaving a 2′-O-methyl (2′-O-ME) substitution to the ribofuranosyl moiety.See Becker et al., “Method for Amplifying Target Nucleic Acids UsingModified Primers,” U.S. Pat. No. 6,130,038. Other sugar modificationsinclude, but are not limited to 2′-amino, 2′-fluoro,(L)-alpha-threofuranosyl, and pentopuranosyl modifications. Thenucleoside subunits may by joined by linkages such as phosphodiesterlinkages, modified linkages or by non-nucleotide moieties which do notprevent hybridization of the oligonucleotide to its complementary targetnucleic acid sequence. Modified linkages include those linkages in whicha standard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine. (DNA analogs having a pseudo peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA” and are disclosed byNielsen et al., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082.) Otherlinkage modifications include, but are not limited to, morpholino bonds.

Non-limiting examples of oligonucleotides or oligomers contemplated bythe present invention include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs (LNAs). See Imanishi etal., “Bicyclonucleoside and Oligonucleotide Analogues,” U.S. Pat. No.6,268,490; and Wengel et al., “Oligonucleotide Analogues,” U.S. Pat. No.6,670,461.) Any nucleic acid analog is contemplated by the presentinvention provided the modified oligonucleotide can perform its intendedfunction, e.g., hybridize to a target nucleic acid under stringenthybridization conditions or amplification conditions, or interact with aDNA or RNA polymerase, thereby initiating extension or transcription. Inthe case of detection probes, the modified oligonucleotides must also becapable of preferentially hybridizing to the target nucleic acid understringent hybridization conditions.

While design and sequence of oligonucleotides for the present inventiondepend on their function as described below, several variables mustgenerally be taken into account. Among the most critical are: length,melting temperature (Tm), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well known aspect ofoligonucleotide design, and various computer programs are readilyavailable to screen large numbers of potential oligonucleotides foroptimal ones.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide is not efficiently extended by theaddition of nucleotides to its 3′-terminus, by a DNA- or RNA-dependentDNA polymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be a “primer.”

As used in this disclosure, the phrase “an oligonucleotide having anucleic acid sequence ‘comprising,’ ‘consisting of’ or ‘consistingessentially of’ a sequence selected from” a group of specific sequencesmeans that the oligonucleotide, as a basic and novel characteristic, iscapable of stably hybridizing to a nucleic acid having the exactcomplement of one of the listed nucleic acid sequences of the groupunder stringent hybridization conditions. An exact complement includesthe corresponding DNA or RNA sequence.

The phrase “an oligonucleotide substantially corresponding to a nucleicacid sequence” means that the referred to oligonucleotide issufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.

One skilled in the art will understand that “substantiallycorresponding” oligonucleotides of the invention can vary from thereferred to sequence and still hybridize to the same target nucleic acidsequence. This variation from the nucleic acid may be stated in terms ofa percentage of identical bases within the sequence or the percentage ofperfectly complementary bases between the probe or primer and its targetsequence. Thus, an oligonucleotide of the present inventionsubstantially corresponds to a reference nucleic acid sequence if thesepercentages of base identity or complementarity are from 100% to about80%. In preferred embodiments, the percentage is from 100% to about 85%.In more preferred embodiments, this percentage can be from 100% to about90%; in other preferred embodiments, this percentage is from 100% toabout 95%. One skilled in the art will understand the variousmodifications to the hybridization conditions that might be required atvarious percentages of complementarity to allow hybridization to aspecific target sequence without causing an unacceptable level ofnon-specific hybridization.

Tagged Oligonucleotide/Heterologous Tag Sequence

A “tagged oligonucleotide” as used herein refers to an oligonucleotidethat comprises at least a first region and a second region, where thefirst region comprises a “target hybridizing sequence” which hybridizesto the 3′-end of a target nucleic acid sequence of interest, and wherethe second region comprises a “tag sequence” situated 5′ to the targethybridizing sequence and which does not stably hybridize or bind to atarget nucleic acid containing the target nucleic acid sequence.Hybridization of the target hybridizing sequence to the target nucleicacid sequence produces a “tagged target nucleic acid sequence.” Thefeatures and design considerations for the target hybridizing sequencecomponent would be the same as for the priming oligonucleotidesdiscussed infra.

The “tag sequence” or “heterologous tag sequence” may be essentially anyheterologous sequence provided that it does not stably hybridize to thetarget nucleic acid sequence of interest and, thereby, participate indetectable amplification. The tag sequence preferably does not stablyhybridize to any sequence derived from the genome of an organism beingtested or, more particularly, to any target nucleic acid under reactionconditions. A tag sequence that is present in a tagged oligonucleotideis preferably designed so as not to substantially impair or interferewith the ability of the target hybridizing sequence to hybridize to itstarget sequence. Moreover, the tag sequence will be of sufficient lengthand composition such that once a complement of the tag sequence has beenincorporated into an initial DNA primer extension product, atag-specific priming oligonucleotide can then be used to participate insubsequent rounds of amplification as described herein. A tag sequenceof the present invention is typically at least 10 nucleotides in length,and may extend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides inlength. Skilled artisans will recognize that the design of tag sequencesand tagged oligonucleotides for use in the present invention can followany of a number of suitable strategies, while still achieving theobjectives and advantages described herein.

In certain embodiments, the tagged oligonucleotide is a “tagged primingoligonucleotide” comprising a tag sequence and a target hybridizingsequence. In other embodiments, the tagged oligonucleotide is a “taggedpromoter oligonucleotide” comprising a tag sequence, a targethybridizing sequence and a promoter sequence situated 5′ to the tagsequence and effective for initiating transcription therefrom.

Inactivating

The term “inactivating” means that a heterologous tag sequence isaltered so that it does not stably bind to a target nucleic acidsequence under amplification conditions. In the case of an unhybridizedtagged oligonucleotide, the term “inactivating” means that the taggedoligonucleotide is altered from an “active” confirmation which permitsthe target hybridizing sequence to hybridize to the target nucleic acidsequence to an “inactive” confirmation which blocks or otherwiseprevents the target hybridizing sequence from hybridizing to the targetnucleic acid sequence. For example, an inactive confirmation may beformed under stringency conditions permitting the tag closing sequenceto form a stable hybrid with the target hybridizing sequence (e.g.,under less stringent conditions than the conditions for forming anactive confirmation of the tagged oligonucleotide). Unless furtheraltered, the tag closing sequence:target hybridizing sequence hybridremains closed under amplification conditions. Alternatively, a duplexformed between the tag closing sequence and the target hybridizingsequence may be altered by an enzyme, such as a DNAse, an S1 nuclease,an endonuclease, such as a restriction enzyme which cleaves adouble-stranded restriction site formed between the tag closing sequenceand the target hybridizing sequence, a ribonuclease activity (e.g.,RNAse H activity) for digesting the RNA component (e.g., targethybridizing sequence) of a DNA:RNA hybrid, or an exonuclease having a3′-to-5′ or 5′-to-3′ activity for removing nucleotides from the targethybridizing sequence hybridized to the tag closing sequence. However, toavoid exposing a sample to a potentially contaminating source of thetarget nucleic acid sequence, the use of enzymes to inactivate taggedoligonucleotides which have not hybridized to the target nucleic acidsequence is generally not preferred. Other inactivating means includechemicals for altering the target hybridizing sequence so that it isincapable of hybridizing to a target nucleic acid sequence underamplification conditions.

Moieties can be included in the tag hybridizing sequence to furtherstabilize hybrids formed between the target closing sequence and thetarget hybridizing sequence of tagged oligonucleotides, especially whereit is anticipated that at least some of the inactive taggedoligonucleotides will be introduced into the amplification reactionmixture. Suitable moieties include modified nucleotides, including LNAs,2′-O-ME ribonucleotides, 2,6 diamino purine. 5-methyl cytosine, and C-5propynyl cytosine or uracil. Those skilled in the art will be able toreadily select the number and positions of such modified nucleotides tolimit breathing at the 5′- and 3′-ends of the tag closing sequence andto achieve a desired melting temperature of the hybrid without engagingundue experimentation. Other suitable moieties include minor groovebinders and pendant groups, such as purine, DABCYL, pyrine and5′-trimethoxy stilbene CAP.

Removing

As used herein, the term “removing” refers to the physical separation oftagged target nucleic acid sequences from unhybridized taggedoligonucleotides. Tagged target nucleic acid sequences can be physicallyseparated from unhybridized tagged oligonucleotides (or heterologous tagsequences) present in a nucleic acid sample by a variety of techniquesknown to those skilled in the art. By way of example, tagged targetnucleic acid sequences can be bound to a solid support and immobilizedin a nucleic acid sample while unbound material is removed. To removeunbound material, the solid support can be subjected to one or morewash/rinse steps. The wash steps are intended to remove remainingunhybridized tagged oligonucleotides and potentially interferingcellular or sample material. A rinse step is typically included wherethe wash solution contains a component that is inhibitory toamplification when present at a sufficiently high concentration, such asa detergent. The solid support preferably binds specifically to targetnucleic acids or tagged target nucleic acid sequences to preventunhybridized tagged oligonucleotide (or unbound heterologous tagsequences) from entering into the amplification reaction. Exemplarymeans for capturing, immobilizing and purifying target nucleic acids arediscussed below, an example of which is disclosed by Weisburg et al.,“Two-Step Hybridization and Capture of a Polynucleotide,” U.S. Pat. No.6,534,273.

Tag Closing Sequence/Tag Closing Oligonucleotide

The phrases “tag closing sequence” and “tag closing oligonucleotide”refer to an oligonucleotide that is complementary to a portion of thetarget hybridizing sequence of a tagged oligonucleotide. The length andsequence of the tag closing sequence are selected so that the tagclosing sequence does not stably hybridize to the target hybridizingsequence of the tagged oligonucleotide under a first set of conditionspermitting stable hybridization of the target hybridizing sequence to atarget sequence. The tag closing sequence may include abasic nucleotidesor base mismatches with the target hybridizing sequence. Provided thetagged oligonucleotide is not hybridized to the target sequence, the tagclosing sequence stably hybridizes to the target hybridizing sequenceunder a second set of less stringent conditions, thus “inactivating” orblocking the tagged oligonucleotide from hybridizing to the targetsequence. The tag closing sequence may be in the form of a discreteoligonucleotide or it may be joined to the 5′-end of a taggedoligonucleotide (“tag molecule”), so that it forms a hairpin structurewith the tagged oligonucleotide under the second set of conditions(“hairpin tag molecule”). If part of a tag molecule, the tag closingsequence is preferably joined to the tagged oligonucleotide via anon-nucleotide linker region (e.g., abasic nucleotides or polyethyleneglycol) of sufficient length for the tag closing sequence to hybridizeto the target hybridizing sequence under the second set of conditions.The tag closing sequence may be modified to prevent the initiation ofDNA synthesis therefrom, which can include a blocking moiety situated atits 3′-terminus. The tag closing sequence is at least 3 but no more thanabout 20 bases in length. Typical tag closing sequences are from 10 to16 bases in length.

Amplification or Nucleic Acid Amplification

By “amplification” or “nucleic acid amplification” is meant productionof multiple copies of a target nucleic acid that contains at least aportion of the intended specific target nucleic acid sequence. Themultiple copies may be referred to as amplicons or amplificationproducts. In certain embodiments, the amplified target contains lessthan the complete target gene sequence (introns and exons) or anexpressed target gene sequence (spliced transcript of exons and flankinguntranslated sequences). For example, specific amplicons may be producedby amplifying a portion of the target polynucleotide by usingamplification primers that hybridize to, and initiate polymerizationfrom, internal positions of the target polynucleotide. Preferably, theamplified portion contains a detectable target sequence that may bedetected using any of a variety of well-known methods.

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (Mullis etal., U.S. Pat. No. 4,683,195; Mullis, U.S. Pat. No. 4,683,202; andMullis et al., U.S. Pat. No. 4,800,159), commonly referred to as PCR,uses multiple cycles of denaturation, annealing of primer pairs toopposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA(Gelfand et al., “Reverse Transcription with Thermostable DNAPolymerases—High Temperature Reverse Transcription,” U.S. Pat. Nos.5,322,770 and 5,310,652). Another method is strand displacementamplification (Walker, G. et al. (1992), Proc. Natl. Acad. Sci. USA 89,392-396; Walker et al., “Nucleic Acid Target Generation,” U.S. Pat. No.5,270,184; Walker, “Strand Displacement Amplification,” U.S. Pat. No.5,455,166; and Walker et al. (1992) Nucleic Acids Research 20,1691-1696), commonly referred to as SDA, which uses cycles of annealingpairs of primer sequences to opposite strands of a target sequence,primer extension in the presence of a dNTP to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (European Pat. No. 0 684 315). Otheramplification methods include: nucleic acid sequence based amplification(Malek et al., U.S. Pat. No. 5,130,238), commonly referred to as NASBA;one that uses an RNA replicase to amplify the probe molecule itself(Lizardi, P. et al. (1988) BioTechnol. 6, 1197-1202), commonly referredto as Qβ replicase; a transcription-based amplification method (Kwoh, D.et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173-1177); self-sustainedsequence replication (Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874-1878; Landgren (1993) Trends in Genetics 9, 199-202; andLee, H. et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)); and,transcription-mediated amplification (Kacian et al., “Nucleic AcidSequence Amplification Methods,” U.S. Pat. No. 5,480,784; and Kacian etal., U.S. Pat. No. 5,399,491), commonly referred to as TMA. For furtherdiscussion of known amplification methods see Persing, David H., 1993,“In Vitro Nucleic Acid Amplification Techniques” in Diagnostic MedicalMicrobiology: Principles and Applications (Persing et al., Eds.), pp.51-87 (American Society for Microbiology, Washington, D.C.). Otherillustrative amplification methods suitable for use in accordance withthe present invention include rolling circle amplification (RCA)(Lizardi, “Rolling Circle Replication Reporter Systems,” U.S. Pat. No.5,854,033); Helicase Dependent Amplification (HDA) (Kong et al.,“Helicase Dependent Amplification Nucleic Acids,” U.S. Pat. Appln. Pub.No. US 2004-0058378 A1); and Loop-Mediated Isothermal Amplification(LAMP) (Notomi et al., “Process for Synthesizing Nucleic Acid,” U.S.Pat. No. 6,410,278).

Preferred transcription-based amplification systems of the presentinvention include TMA, which employs an RNA polymerase to producemultiple RNA transcripts of a target region (e.g., Kacian et al., U.S.Pat. Nos. 5,480,784 and 5,399,491; and Becker et al., “Single-PrimerNucleic Acid Amplification Methods,” U.S. Pat. Appln. Pub. No. US2006-0046265 A1). TMA uses a “promoter oligonucleotide” or“promoter-primer” that hybridizes to a target nucleic acid in thepresence of a reverse transcriptase and an RNA polymerase to form adouble-stranded promoter from which the RNA polymerase produces RNAtranscripts. These transcripts can become templates for further roundsof TMA in the presence of a second primer capable of hybridizing to theRNA transcripts. Unlike PCR, LCR or other methods that require heatdenaturation, TMA is an isothermal method that uses an RNAse H activityto digest the RNA strand of an RNA:DNA hybrid, thereby making the DNAstrand available for hybridization with a primer or promoter-primer.Generally, the RNAse H activity associated with the reversetranscriptase provided for amplification is used.

In one illustrative TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce primer extension. From an unmodifiedpromoter-primer, reverse transcriptase creates a cDNA copy of the targetRNA, while RNAse H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer”. From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly-synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNAse H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons. Thus, a billion-fold isothermic amplification can be achievedusing two amplification primers.

In another illustrative TMA method, one or more features as described inBecker et al., U.S. Pat. Appln. Pub. No. US 2006-0046265 A1 areoptionally incorporated. Preferred TMA methods in this respect includethe use of blocking moieties, terminating moieties, and other modifyingmoieties that provide improved TMA process sensitivity and accuracy.Thus, certain preferred embodiments of the present invention employtagged oligonucleotides, as described herein, in conjunction with themethods as described in Becker et al., U.S. Pat. Appln. Pub. No. US2006-0046265 A1.

By “detectable amplification” is meant that a detectable signalassociated with an amplification product in an amplification reactionmixture rises above a predetermined background or threshold level(end-point amplification) or rises above a background or threshold levelwithin a predetermined period of time (real-time amplification). See,e.g., Light et al., “Method for Determining the Amount of an Analyte ina Sample,” U.S. Pat. Appln. Pub. No. US 2006-0276972, paragraphs506-549. The amplification product contains a sequence having sequenceidentity with a target nucleic acid sequence or its complement and canbe detected with, for example, an intercalating dye or a detection probehaving specificity for a region of the target nucleic acid sequence orits complement.

“Selective Amplification”

“Selective amplification” as used herein, refers to the amplification ofa target nucleic acid sequence according to the present invention wheredetectable amplification of the target sequence is limited orsubstantially limited to amplification of target sequence contributed bysample of interest that is being tested and is not contributed by targetnucleic acid sequence contributed by some other sample source, e.g.,contamination present in reagents or components used duringamplification reactions or in the environment or environmentalconditions in which amplification reactions are performed.

Amplification Conditions

By “amplification conditions” is meant conditions permitting nucleicacid amplification according to the present invention. Amplificationconditions may, in some embodiments, be less stringent than “stringenthybridization conditions” as described herein. Oligonucleotides used inthe amplification reactions of the present invention hybridize to theirintended targets under amplification conditions, but may or may nothybridize under stringent hybridization conditions. On the other hand,detection probes of the present invention hybridize under stringenthybridization conditions. While the Examples section infra providespreferred amplification conditions for amplifying target nucleic acidsequences according to the present invention, other acceptableconditions to carry out nucleic acid amplifications according to thepresent invention could be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

Hybridize/Hybridization

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof. The two constituentstrands of this double-stranded structure, sometimes called a hybrid,are held together by hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can also form between baseswhich are not members of these “canonical” pairs. Non-canonical basepairing is well-known in the art. (See, e.g., ROGER L. P. ADAMS ET AL.,THE BIOCHEMISTRY OF THE NUCLEIC ACIDS (11^(th) ed. 1992).)

“Stringent hybridization conditions” or “stringent conditions” refer toconditions where a specific detection probe is able to hybridize withtarget nucleic acids over other nucleic acids present in the testsample. It will be appreciated that these conditions may vary dependingupon factors including the GC content and length of the probe, thehybridization temperature, the composition of the hybridization reagentor solution, and the degree of hybridization specificity sought.Specific stringent hybridization conditions are provided in thedisclosure below.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereeach strand is complementary to the other, and where the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in astable, double-stranded hydrogen-bonded region under stringenthybridization or amplification conditions. When a contiguous sequence ofnucleotides of one single-stranded region is able to form a series of“canonical” hydrogen-bonded base pairs with an analogous sequence ofnucleotides of the other single-stranded region, such that A is pairedwith U or T and C is paired with G, the nucleotides sequences are“perfectly” complementary.

By “preferentially hybridize” is meant that under stringenthybridization conditions, certain complementary nucleotides ornucleobase sequences hybridize to form a stable hybrid preferentiallyover other, less stable duplexes. By “does not stably hybridize” ismeant that a stable hybrid is not formed in appreciable and/ordetectable amounts under a defined set of conditions.

By “stable” or “stably hybridize” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

Promoter Oligonucleotide/Promoter Sequence

As is well known in the art, a “promoter” is a specific nucleic acidsequence that is recognized by a DNA-dependent RNA polymerase(“transcriptase”) as a signal to bind to the nucleic acid and begin thetranscription of RNA at a specific site. For binding, it was generallythought that such transcriptases required DNA which had been rendereddouble-stranded in the region comprising the promoter sequence via anextension reaction, however, the present inventors have determined thatefficient transcription of RNA can take place even under conditionswhere a double-stranded promoter is not formed through an extensionreaction with the template nucleic acid. The template nucleic acid (thesequence to be transcribed) need not be double-stranded. IndividualDNA-dependent RNA polymerases recognize a variety of different promotersequences, which can vary markedly in their efficiency in promotingtranscription. When an RNA polymerase binds to a promoter sequence toinitiate transcription, that promoter sequence is not part of thesequence transcribed. Thus, the RNA transcripts produced thereby willnot include that sequence.

According to the present invention, a “promoter oligonucleotide” refersto an oligonucleotide comprising first and second regions, and which ispreferably modified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter oligonucleotide of thepresent invention comprises a base sequence which hybridizes to a DNAtemplate, where the hybridizing sequence is situated 3′, but notnecessarily adjacent to, a promoter region. The hybridizing portion of apromoter oligonucleotide of the present invention is typically at least10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. The “second region” comprises apromoter for an RNA polymerase. A promoter oligonucleotide of thepresent invention is engineered so that it is incapable of beingextended by an RNA- or DNA-dependent DNA polymerase, e.g., reversetranscriptase, preferably comprising a blocking moiety at its3′-terminus as described above. Suitable and preferred promoteroligonucleotides are described herein.

Universal/Pan Oligonucleotides

“Universal” oligonucleotides or “pan” oligonucleotides includeoligonucleotides that can be used in an amplification reaction toidentify the presence of nucleic acid sequences of a class of organismsbased upon highly conserved sequences that are unique to a class oforganisms. (As used herein, the term “class” does not necessarily implya recognized phylogenetic grouping or organisms.) For example, thehighly conserved 16S ribosomal RNA-coding sequences contain regions thatare found in bacteria, or groupings of bacteria (e.g., Eubacteria,Gram-positive bacteria or Gram-negative bacteria), but are not in humansand other higher organisms, and thus oligonucleotides may be designedand used in a nucleic acid amplification reaction to detect the presenceof bacterial sequences in a sample of interest. See, e.g., McCabe et al.(1999) Molecular Genetics and Metabolism 66, 205-211; Schmidt, T. et al.(1994) Meth. Enzymol. 235, 205-222 (method for identifying pathogens);Kunishima, S. et al., (2000) Transfusion 40, 1420 (method for detectingbacteria in blood); Greisen, K. (1994) J. Clin. Microbiol. 32, 335-351(method for detecting pathogenic bacteria in cerebral spinal fluid);Jordan, J. (2005) J. Mol. Diag. 7, 575-581 (method for diagnosing sepsisin neonates); Rothman, R. et al. (2002) J. Infect. Dis. 186, 1677-1681(method for diagnosing acute bacterial endocarditis); and Cox, C. et al.(2002) Arthritis Res. Ther. 5, R1-R8 (detecting bacteria in synovialfluid). Similarly, universal oligonucleotides for other classes oforganisms, such as fungal pathogens, have been described. See, e.g.,Maaroati, Y. et al. (2003) J. Clin. Microbiol. 41, 3293-3298 (method forquantifying Candida albicans in blood); Carr, M. et al. (2005) J. Clin.Microbiol. 43, 3023-3026 (method for detecting Candida dubliniensis inblood); and White, P. et al. (2003) J. Med. Microbiol. 52, 229-238(method for detecting systemic fungal infections). Essentially anyuniversal oligonucleotides known or developed for a given class oforganism may be advantageously employed in the methods described herein.

Priming Oligonucleotide

A priming oligonucleotide is an oligonucleotide, at least the 3′-end ofwhich is complementary to a nucleic acid template, and which complexes(by hydrogen bonding or hybridization) with the template to give aprimer:template complex suitable for initiation of synthesis by an RNA-or DNA-dependent DNA polymerase. A priming oligonucleotide is extendedby the addition of covalently bonded nucleotide bases to its3′-terminus, which bases are complementary to the template. The resultis a primer extension product. A priming oligonucleotide of the presentinvention is typically at least 10 nucleotides in length, and may extendup to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Suitableand preferred priming oligonucleotides are described herein. Virtuallyall DNA polymerases (including reverse transcriptases) that are knownrequire complexing of an oligonucleotide to a single-stranded template(“priming”) to initiate DNA synthesis, whereas RNA replication andtranscription (copying of RNA from DNA) generally do not require aprimer. By its very nature of being extended by a DNA polymerase, apriming oligonucleotide does not comprise a 3′-blocking moiety.

Displacer Oligonucleotide

A “displacer oligonucleotide” is a priming oligonucleotide whichhybridizes to a template nucleic acid upstream from a neighboringpriming oligonucleotide hybridized to the 3′-end of a target sequence(referred to herein as the “forward priming oligonucleotide”). By“upstream” is meant that a 3′-end of the displacer oligonucleotidecomplexes with the template nucleic acid 5′ to a 3′-end of the forwardpriming oligonucleotide. When hybridized to the template nucleic acid,the 3′-terminal base of the displacer oligonucleotide is preferablyadjacent to or spaced from the 5-terminal base of the forward primingoligonucleotide. More preferably, the 3′-terminal base of the displaceroligonucleotide is spaced from 5 to 35 bases from the 5′-terminal baseof the forward priming oligonucleotide. The displacer oligonucleotidemay be provided to a reaction mixture contemporaneously with the forwardpriming oligonucleotide or after the forward priming oligonucleotide hashad sufficient time to hybridize to the template nucleic acid. Extensionof the forward priming oligonucleotide can be initiated prior to orafter the displacer oligonucleotide is provided to a reaction mixture.Under amplification conditions, the displacer oligonucleotide isextended in a template-dependent manner, thereby displacing a primerextension product comprising the forward priming oligonucleotide whichis complexed with the template nucleic acid. Once displaced from thetemplate nucleic acid, the primer extension product comprising theforward priming oligonucleotide is available for complexing with apromoter oligonucleotide. The forward priming oligonucleotide and thedisplacer oligonucleotide both preferentially hybridize to the targetnucleic acid. Examples of displacer oligonucleotides and their uses aredisclosed by Becker et al., “Methods and Kits for Amplifying DNA,” U.S.Pat. No. 7,713,697, which enjoys common ownership herewith.

Blocking Moiety

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. A blockingmoiety may be a small molecule, e.g., a phosphate or ammonium group, orit may be a modified nucleotide, e.g., a 3′2′ dideoxynucleotide or 3′deoxyadenosine 5′-triphosphate (cordycepin), or other modifiednucleotide. Additional blocking moieties include, for example, the useof a nucleotide or a short nucleotide sequence having a 3′-to-5′orientation, so that there is no free hydroxyl group at the 3′-terminus,the use of a 3′ alkyl group, a 3′ non-nucleotide moiety (see, e.g.,Arnold et al., “Non-Nucleotide Linking Reagents for Nucleotide Probes,”U.S. Pat. No. 6,031,091), phosphorothioate, alkane-diol residues,peptide nucleic acid (PNA), nucleotide residues lacking a 3′ hydroxylgroup at the 3′-terminus, or a nucleic acid binding protein. Preferably,the 3′-blocking moiety comprises a nucleotide or a nucleotide sequencehaving a 3′-to-5′ orientation or a 3′ non-nucleotide moiety, and not a3′2′-dideoxynucleotide or a 3′ terminus having a free hydroxyl group.Additional methods to prepare 3′-blocking oligonucleotides are wellknown to those of ordinary skill in the art.

Binding Molecule

As used herein, a “binding molecule” is a substance which hybridizes toor otherwise binds to an RNA target nucleic acid adjacent to or near the5′-end of the desired target sequence, so as to limit a DNA primerextension product to a desired length, i.e., a primer extension producthaving a generally defined 3′-end. As used herein, the phrase “defined3′-end” means that the 3′-end of a primer extension product is notwholly indeterminate, as would be the case in a primer extensionreaction which occurs in the absence of a binding molecule, but ratherthat the 3′-end of the primer extension product is generally known towithin a small range of bases. In certain embodiments, a bindingmolecule comprises a base region. The base region may be DNA, RNA, aDNA:RNA chimeric molecule, or an analog thereof. Binding moleculescomprising a base region may be modified in one or more ways, asdescribed herein. Exemplary base regions include terminating anddigestion oligonucleotides, as described below. In other embodiments, abinding molecule may comprise, for example, a protein or drug capable ofbinding RNA with sufficient affinity and specificity to limit a DNAprimer extension product to a pre-determined length.

Terminating Oligonucleotide

In the present invention, a “terminating oligonucleotide” is anoligonucleotide comprising a base sequence that is complementary to aregion of the target nucleic acid in the vicinity of the 5′-end of thetarget sequence, so as to “terminate” primer extension of a nascentnucleic acid that includes a priming oligonucleotide, thereby providinga defined 3′-end for the nascent nucleic acid strand. A terminatingoligonucleotide is designed to hybridize to the target nucleic acid at aposition sufficient to achieve the desired 3′-end for the nascentnucleic acid strand. The positioning of the terminating oligonucleotideis flexible depending upon its design. A terminating oligonucleotide maybe modified or unmodified. In certain embodiments, terminatingoligonucleotides are synthesized with at least one or more 2′-O-MEribonucleotides. These modified nucleotides have demonstrated higherthermal stability of complementary duplexes. The 2′-O-ME ribonucleotidesalso function to increase the resistance of oligonucleotides toexonucleases, thereby increasing the half-life of the modifiedoligonucleotides. See, e.g., Majlessi et al. (1988) Nucleic Acids Res.26, 2224-9. Other modifications as described elsewhere herein may beutilized in addition to or in place of 2′-O-ME ribonucleotides. Forexample, a terminating oligonucleotide may comprise PNA or an LNA. See,e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53. A terminatingoligonucleotide of the present invention typically includes a blockingmoiety at its 3′-terminus to prevent extension. A terminatingoligonucleotide may also comprise a protein or peptide joined to theoligonucleotide so as to terminate further extension of a nascentnucleic acid chain by a polymerase. A terminating oligonucleotide of thepresent invention is typically at least 10 bases in length, and mayextend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length.Suitable and preferred terminating oligonucleotides are describedherein. It should be noted that while a terminating oligonucleotidetypically or necessarily includes a 3′-blocking moiety, “3′-blocked”oligonucleotides are not necessarily terminating oligonucleotides. Otheroligonucleotides of the present invention, e.g., promoteroligonucleotides and capping oligonucleotides are typically ornecessarily 3′-blocked as well.

Insertion Sequence

As used herein, an “insertion sequence” is a sequence positioned betweenthe first region (i.e., template binding portion) and the second regionof a promoter oligonucleotide. Insertion sequences are preferably 5 to20 nucleotides in length, more preferably 6 to 18 nucleotides in length,and most preferably 6 to 12 nucleotides in length. The inclusion ofinsertion sequences in promoter oligonucleotides increases the rate atwhich RNA amplification products are formed. Exemplary insertionsequences are described herein.

Target Capture

Target capture, as used herein, includes any technique effective toremove all or substantially all unhybridized tagged oligonucleotideafter hybridization of tagged oligonucleotide with a target nucleic acidsequence but prior to amplification of the target nucleic acid sequence.Generally, target capture involves capturing a target polynucleotideonto a solid support, such as magnetically attractable particles, wherethe solid support retains the target polynucleotide during one or morewashing steps of the target polynucleotide purification procedure. Inthis way, a target polynucleotide is substantially purified fromunhybridized tagged oligonucleotide prior to a subsequent nucleic acidamplification step. Numerous target capture methods are known andsuitable for use in conjunction with the methods described herein.

For example, one illustrative approach described in U.S. Pat. Appln.Pub. No. US 2006-0068417 A1 uses at least one capture probeoligonucleotide that contains a target-complementary region and a memberof a specific binding pair that joins a target nucleic acid to animmobilized probe on a capture support, thus forming a capture hybridthat is separated from other sample components of a sample. In anotherillustrative method, Weisburg et al., in U.S. Pat. No. 6,110,678,describe a method for capturing a target polynucleotide in a sample ontoa solid support, such as magnetically attractable particles, with anattached immobilized probe by using a capture probe and two differenthybridization conditions, which preferably differ in temperature only.The two hybridization conditions control the order of hybridization,where the first hybridization conditions allow hybridization of thecapture probe to the target polynucleotide, and the second hybridizationconditions allow hybridization of the capture probe to the immobilizedprobe. The method may be used to detect the presence of a targetpolynucleotide in a sample by detecting the captured targetpolynucleotide or amplified target polynucleotide.

Another illustrative target capture technique involves a hybridizationsandwich technique for capturing and for detecting the presence of atarget polynucleotide. See Ranki et al., “Detection of Microbial NucleicAcids By a One-Step Sandwich Hybridization Test,” U.S. Pat. No.4,486,539. The technique involves the capture of the targetpolynucleotide by a probe bound to a solid support and hybridization ofa detection probe to the captured target polynucleotide. Detectionprobes not hybridized to the target polynucleotide are readily washedaway from the solid support. Thus, remaining label is associated withthe target polynucleotide initially present in the sample.

Another illustrative target capture technique involves a method thatuses a mediator polynucleotide that hybridizes to both a targetpolynucleotide and to a polynucleotide fixed on a solid support. SeeStabinsky, “Methods and Kits for Performing Nucleic Acid HybridizationAssays,” U.S. Pat. No. 4,751,177. The mediator polynucleotide joins thetarget polynucleotide to the solid support to produce a bound target. Alabeled probe can be hybridized to the bound target and unbound labeledpro can be washed away from the solid support.

Yet another illustrative target capture technique is disclosed byEnglelhardt, “Capture Sandwich Hybridization Method and Composition,”U.S. Pat. No. 5,288,609, which describes a method for detecting a targetpolynucleotide. The method utilizes two single-stranded polynucleotidesegments complementary to the same or opposite strands of the target andresults in the formation of a double hybrid with the targetpolynucleotide. In one embodiment, the hybrid is captured onto asupport.

In another illustrative target capture technique, methods and kits fordetecting nucleic acids use oligonucleotide primers labeled withspecific binding partners to immobilize primers and primer extensionproducts. See Burdick et al., “Diagnostic Kit and Method Using a SolidPhase Capture Means for Detecting Nucleic Acids,” European Pat. Appln.No. 0 370 694 A2. The label specifically complexes with its receptorwhich is bound to a solid support.

The above capture techniques are illustrative only, and not limiting.Indeed, essentially any technique available to the skilled artisan maybe used provided it is effective for removing all or substantially allunhybridized tagged oligonucleotide after hybridization of taggedoligonucleotide with a target nucleic acid sequence but prior toamplification of the target nucleic acid sequence, as described herein.

Probe

By “probe” or “detection probe” is meant a molecule comprising anoligonucleotide having a base sequence partly or completelycomplementary to a region of a target sequence sought to be detected, soas to hybridize thereto under stringent hybridization conditions. Aswould be understood by someone having ordinary skill in the art, a probecomprises an isolated nucleic acid molecule, or an analog thereof, in aform not found in nature without human intervention (e.g., recombinedwith foreign nucleic acid, isolated, or purified to some extent).

The probes of this invention may have additional nucleosides ornucleobases outside of the targeted region so long as such nucleosidesor nucleobases do not substantially affect hybridization under stringenthybridization conditions and, in the case of detection probes, do notprevent preferential hybridization to the target nucleic acid. Anon-complementary sequence may also be included, such as a targetcapture sequence (generally a homopolymer tract, such as a poly-A,poly-T or poly-U tail), promoter sequence, a binding site for RNAtranscription, a restriction endonuclease recognition site, or maycontain sequences which will confer a desired secondary or tertiarystructure, such as a catalytic active site or a hairpin structure on theprobe, on the target nucleic acid, or both.

The probes preferably include at least one detectable label. The labelmay be any suitable labeling substance, including but not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye,a hapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a base sequence region that is unable to stably hybridizeto the target nucleic acid under the stated conditions, and mixtures ofthese. In one particularly preferred embodiment, the label is anacridinium ester. Probes may also include interacting labels which emitdifferent signals, depending on whether the probes have hybridized totarget sequences. Examples of interacting labels includeenzyme/substrates, enzyme/cofactor, luminescent/quencher,luminescent/adduct, dye dimers, and Förrester energy transfer pairs.Certain probes of the present invention do not include a label. Forexample, non-labeled “capture” probes may be used to enrich for targetsequences or replicates thereof, which may then be detected by a second“detection” probe. See, e.g., Weisburg et al., U.S. Pat. No. 6,534,273.While detection probes are typically labeled, certain detectiontechnologies do not require that the probe be labeled. See, e.g., Nygrenet al., “Devices and Methods for Optical Detection of Nucleic AcidHybridization,” U.S. Pat. No. 6,060,237.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is morepreferably at least 5° C. below the melting temperature of the nucleicacid duplex, and even more preferably at least 10° C. below the meltingtemperature of the reaction mixture.

By “preferentially hybridize” is meant that under stringenthybridization conditions, probes of the present invention hybridize totheir target sequences, or replicates thereof, to form stableprobe:target hybrids, while at the same time formation of stableprobe:non-target hybrids is minimized. Thus, a probe hybridizes to atarget sequence or replicate thereof to a sufficiently greater extentthan to a non-target sequence, to enable one having ordinary skill inthe art to accurately quantitate the RNA replicates or complementary DNA(cDNA) of the target sequence formed during the amplification.

Probes of a defined sequence may be produced by techniques known tothose of ordinary skill in the art, such as by chemical synthesis, andby in vitro or in vivo expression from recombinant nucleic acidmolecules. Preferably probes are 10 to 100 nucleotides in length, morepreferably 12 to 50 bases in length, and even more preferably 18 to 35bases in length.

Nucleic Acid “Identity”

In certain embodiments, a nucleic acid of the present inventioncomprises a contiguous base region that is at least 80%, 90%, or 100%identical to a contiguous base region of a reference nucleic acid. Forshort nucleic acids, e.g., certain oligonucleotides of the presentinvention, the degree of identity between a base region of a “query”nucleic acid and a base region of a reference nucleic acid can bedetermined by manual alignment. “Identity” is determined by comparingjust the sequence of nitrogenous bases, irrespective of the sugar andbackbone regions of the nucleic acids being compared. Thus, thequery:reference base sequence alignment may be DNA:DNA, RNA:RNA,DNA:RNA, RNA:DNA, or any combinations or analogs thereof. Equivalent RNAand DNA base sequences can be compared by converting U's (in RNA) to T's(in DNA).

Template

A “template” is a nucleic acid molecule that is being copied by anucleic acid polymerase. A template may be single-stranded,double-stranded or partially double-stranded, depending on thepolymerase. The synthesized copy is complementary to the template or toat least one strand of a double-stranded or partially double-strandedtemplate. Both RNA and DNA are typically synthesized in the 5′-to-3′direction and the two strands of a nucleic acid duplex are aligned sothat the 5′-termini of the two strands are at opposite ends of theduplex (and, by necessity, so then are the 3′-termini). While accordingto the present invention, a “target sequence” is always a “template,”templates can also include secondary primer extension products andamplification products.

DNA-dependent DNA Polymerase

A “DNA-dependent DNA polymerase” is an enzyme that synthesizes acomplementary DNA copy from a DNA template. Examples are Taq DNApolymerase, a highly thermostable DNA polymerase from the thermophilicbacterium Therms aquaticus, for PCR amplification reactions, DNApolymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases of the present invention may be the naturally occurringenzymes isolated from bacteria or bacteriophages or expressedrecombinantly, or may be modified or “evolved” forms which have beenengineered to possess certain desirable characteristics, e.g.,thermostability, or the ability to recognize or synthesize a DNA strandfrom various modified templates. All known DNA-dependent DNA polymerasesrequire a complementary primer to initiate synthesis. It is known thatunder suitable conditions a DNA-dependent DNA polymerase may synthesizea complementary DNA copy from an RNA template. RNA-dependent DNApolymerases (described below) typically also have DNA-dependent DNApolymerase activity. An example of such a polymerase is the MasterAmp™Tth DNA Polymerase, which has both DNA-dependent and RNA-dependent(i.e., reverse transcriptase) DNA polymerase activities that can be usedin both PCR and RT-PCR amplification reactions (EpicentreBiotechnologies, Madison, Wis.).

DNA-dependent RNA Polymerase (Transcriptase)

A “DNA-dependent RNA polymerase” or “transcriptase” is an enzyme thatsynthesizes multiple RNA copies from a double-stranded orpartially-double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

RNA-dependent DNA Polymerase (Reverse Transcriptase)

An “RNA-dependent DNA polymerase” or “reverse transcriptase” (“RT”) isan enzyme that synthesizes a complementary DNA copy from an RNAtemplate. All known reverse transcriptases also have the ability to makea complementary DNA copy from a DNA template; thus, they are both RNA-and DNA-dependent DNA polymerases. RTs may also have an RNAse Hactivity. Preferred is reverse transcriptase derived from Maloney murineleukemia virus (MMLV-RT). A primer is required to initiate synthesiswith both RNA and DNA templates.

Selective RNAses

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes other than RNAse H which possess the same or similar activityare also contemplated in the present invention. Selective RNAses may beendonucleases or exonucleases. Most reverse transcriptase enzymescontain an RNAse H activity in addition to their polymerase activities.However, other sources of the RNAse H are available without anassociated polymerase activity. The degradation may result in separationof RNA from a RNA:DNA complex. Alternatively, a selective RNAse maysimply cut the RNA at various locations such that portions of the RNAmelt off or permit enzymes to unwind portions of the RNA. Other enzymeswhich selectively degrade RNA target sequences or RNA products of thepresent invention will be readily apparent to those of ordinary skill inthe art.

Sense/Antisense Strand(s)

Discussions of nucleic acid synthesis are greatly simplified andclarified by adopting terms to name the two complementary strands of anucleic acid duplex. Traditionally, the strand encoding the sequencesused to produce proteins or structural RNAs are designated as the “sense(+)” strand and its complement the “antisense (−)” strand. It is nowknown that in many cases, both strands are functional, and theassignment of the designation “sense” to one and “antisense” to theother must then be arbitrary. Nevertheless, the terms are very usefulfor designating the sequence orientation of nucleic acids and will beemployed herein for that purpose.

Specificity of the System

The term “specificity,” in the context of an amplification system, isused herein to refer to the characteristic of an amplification systemwhich describes its ability to distinguish between target and non-targetsequences dependent on sequence and assay conditions. In terms of anucleic acid amplification, specificity generally refers to the ratio ofthe number of specific amplicons produced to the number of side-products(i.e., the signal-to-noise ratio), described in more detail below.

Sensitivity

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, i.e., the ratio of specific amplicons toside-products.

Bioprocess

A “bioprocess,” as used herein, refers generally to any process in whichliving cells, or components thereof, are present, either intended orunintended. For example, essentially any manufacturing or other processthat employs one or more samples or sample streams, at least one ofwhich comprises living cells, or components thereof, or may comprisesuch cells or components as a result of unintended contamination, isconsidered a bioprocess. In many such processes it is desirable to havethe ability to detect, identify and/or control the presence and/orsources of living cells or components thereof within a process. Usingthe methods of the present invention, for example, the presence and/orsources of contaminating microorganisms or other biological material orcomponents thereof in one or more bioprocess samples or streams may bemonitored. In addition, the purification/sterilization requirementswithin certain samples/streams of a bioprocess may be advantageouslyreduced using the methods of the invention as set forth herein.

As discussed above, the present invention is directed generally tonucleic acid amplification methods that desirably reduce or eliminatefalse positive amplification signals resulting from contaminatingbiological material, such as nucleic acid material, that may be presentin one or more reagents, samples or components that are used in anamplification reaction, or that may be present in the environment inwhich amplification reactions are performed. The invention furtheroffers the advantage of requiring less stringent purification and/orsterility efforts conventionally needed in order to ensure that enzymesand other reagents used in amplification reactions, and the environmentin which amplification reactions are performed, are free of bacterialand other nucleic acid contamination that may yield false positiveresults. Accordingly, the methods of the invention are particularlyuseful in detecting, monitoring and/or quantitating microorganisms (orcontaminating nucleic acids from other sources) in clinical samples,bioprocess samples or sample streams, foodstuffs, water, industrial andenvironmental samples, seed stocks, and other types of material wherethe presence of microorganisms or other forms of contamination may needto be detected and/or monitored.

The present invention can be adapted for use in essentially anyamplification procedure requiring a template-binding primingoligonucleotide capable of extension in the presence of nucleic acidpolymerase. Incorporation of the tagged oligonucleotides (orheterologous tag sequences) into such primer-dependent amplificationprocedures can be accomplished without substantially modifying thereagents and reaction conditions of such procedures. Any neededmodifications should be minor and would be well within the knowledge andcapabilities of a skilled molecular biologist. Descriptions of variousillustrative amplification procedures adopting tagged oligonucleotidesfollows.

FIG. 1 illustrates an adaptation of an isothermal, transcription-basedamplification reaction known as reverse transcription-mediatedamplification (rTMA), various aspects of which are disclosed in Beckeret al., U.S. Pat. No. 7,374,885. The reaction of this illustrativeembodiment is initiated by treating an RNA target sequence in a nucleicacid sample with both a tagged priming oligonucleotide and a terminatingoligonucleotide. The tagged priming oligonucleotide includes a targethybridizing sequence that hybridizes to a 3′-end of the target sequenceand a tag sequence situated 5′ to the target hybridizing sequence. Theterminating oligonucleotide hybridizes to a target nucleic acidcontaining the target sequence in the vicinity of the 5′-end of thetarget sequence. The terminating oligonucleotide is used to end primerextension of a nascent nucleic acid that includes the tagged primingoligonucleotide. Thus, the target nucleic acid forms a stable complexwith the tagged priming oligonucleotide at the 3′-end of the targetsequence and the terminating oligonucleotide located adjacent to or nearthe 5′-end of the target sequence prior to initiating a primer extensionreaction. See FIG. 1, Step 1. Unhybridized tagged primingoligonucleotide is made unavailable for hybridization to the targetsequence prior to initiating a primer extension reaction with the taggedpriming oligonucleotide, preferably by inactivating and/or removing theunhybridized tagged priming oligonucleotide from the nucleic acidsample.

An extension reaction is then initiated from the 3′-end of the taggedpriming oligonucleotide with a DNA polymerase, e.g., reversetranscriptase, to produce a first DNA primer extension product thatincludes the tag sequence and a region complementary to the targetsequence. See FIG. 1, Steps 2 and 3. The first DNA primer extensionproduct is then separated from the target sequence using an enzyme thatselectively degrades the target sequence (e.g., RNAse H activity). SeeFIG. 1, Step 4.

Next, the first DNA primer extension product is treated with a promoteroligonucleotide having a hybridizing sequence and a promoter for an RNApolymerase situated 5′ to the hybridizing sequence. The hybridizingsequence hybridizes to a region of the first DNA primer extensionproduct that is complementary to the 3′-end of the target sequence,thereby forming a promoter oligonucleotide:first DNA primer extensionproduct hybrid. In the illustrated reaction, the promoteroligonucleotide is modified to prevent the initiation of DNA synthesis,preferably by situating a blocking moiety at the 3′-end of the promoteroligonucleotide (e.g., nucleotide sequence having a 3′-to-5′orientation). See FIG. 1, Step 5. The 3′-end of the first DNA primerextension product is preferably extended to add a sequence complementaryto the promoter, resulting in the formation of a double-strandedpromoter sequence. See FIG. 1, Steps 6 and 7. Multiple copies of a firstRNA product complementary to at least a portion of the first DNA primerextension product, not including the promoter portion, are thentranscribed using an RNA polymerase which recognizes the double-strandedpromoter and initiates transcription therefrom. See FIG. 1, Steps 8 and9. As a result, the base sequence of the first RNA product issubstantially identical to the base sequence of the target sequence andthe complement of the tag sequence.

The first RNA products are treated with a priming oligonucleotide whichhybridizes to the complement of the tag sequence to form a primingoligonucleotide:first RNA product hybrid, and the 3′-end of the primingoligonucleotide is extended with the DNA polymerase to produce a secondDNA primer extension product complementary to the first RNA product. SeeFIG. 1, Steps 10-12. The second DNA primer extension product is thenseparated from the first RNA product using an enzyme that selectivelydegrades the first RNA product (e.g., RNAse H activity). See FIG. 1,Step 13.

The second DNA primer extension product is treated with the promoteroligonucleotide, which hybridizes to the 3′-end of the second DNA primerextension product to form a promoter oligonucleotide:second DNA primerextension product hybrid. See FIG. 1, Step 14. The promoteroligonucleotide:second DNA primer extension product hybrid thenre-enters the amplification cycle at Step 6 of FIG. 1, wheretranscription is initiated from the double-stranded promoter and thecycle continues.

FIG. 3 illustrates an adaptation of an isothermal, transcription-basedamplification reaction referred to as transcription-mediatedamplification (TMA), various aspects of which are disclosed in Kacian etal., U.S. Pat. Nos. 5,399,491 and 5,824,518. The reaction of thisillustrative embodiment is initiated by treating an RNA target sequencein a nucleic acid sample with a tagged promoter oligonucleotide. Thetagged promoter oligonucleotide includes a tag sequence, a targethybridizing sequence and a promoter sequence for an RNA polymerase,where the target hybridizing sequence hybridizes to a 3′-end of thetarget sequence. Thus, the target sequence forms a stable complex withthe tagged promoter oligonucleotide at the 3′-end of the target sequenceprior to initiating a primer extension reaction. See FIG. 3, Step 1. Thepromoter sequence is situated 5′ to the tag sequence, and the tagsequence is situated 5′ to the target hybridizing sequence. Unhybridizedtagged promoter oligonucleotide is made unavailable for hybridization tothe target sequence prior to initiating a primer extension reaction withthe tagged priming oligonucleotide, preferably by inactivating and/orremoving the unhybridized tagged priming oligonucleotide from thenucleic acid sample.

An extension reaction is then initiated from the 3′-end of the taggedpromoter oligonucleotide with a DNA polymerase, e.g., reversetranscriptase, to produce a first DNA primer extension product thatincludes the tag and promoter sequence and a region complementary to thetarget sequence. See FIG. 1, Steps 2 and 3. The first DNA primerextension product is then separated from the target sequence to which itis hybridized using an enzyme that selectively degrades that portion ofthe target sequence which is hybridized to the first DNA primerextension product (e.g., RNAse H activity). See FIG. 3, Step 4.

Next, the first DNA primer extension product is treated with a primingoligonucleotide which hybridizes to a region of the first DNA primerextension product that is complementary to a 5′-end of the targetsequence, thereby forming a priming oligonucleotide:first DNA primerextension product hybrid. See FIG. 3, Step 5. The 3′-end of the primingoligonucleotide is extended by a DNA polymerase to produce a second DNAprimer extension product complementary to at least a portion of thefirst DNA primer extension product, and containing a double-strandedpromoter sequence. See FIG. 3, Steps 6 and 7. This second DNA primerextension product is used as a template to transcribe multiple copies ofa first RNA product complementary to the second DNA primer extensionproduct, not including the promoter portion, using an RNA polymerasewhich recognizes the double-stranded promoter and initiatestranscription therefrom. See FIG. 3, Step 8 and 9. The base sequence ofthe first RNA product is substantially identical to the base sequence ofthe tag sequence and the complement of the target sequence.

The first RNA product is treated with the priming oligonucleotide, the3′-end of which is extended by the DNA polymerase to produce a third DNAprimer extension product complementary to the first RNA product. SeeFIG. 3, Steps 10-12. The third DNA primer extension product is thenseparated from the first RNA product using an enzyme which selectivelydegrades the first RNA product (e.g., RNAse H activity). See FIG. 3,Step 13. The third DNA primer extension product is treated with apromoter oligonucleotide having a hybridizing sequence which hybridizesto a complement of the tag sequence at the 3′-end of the third DNAprimer extension product, and further comprises a promoter for an RNApolymerase which is situated 5′ to the hybridizing sequence. See FIG. 3,Step 14. The 3′-end of the third DNA primer extension product isextended to add sequence complementary to the promoter sequence. SeeFIG. 3, Step 15. The 3′-end of the promoter oligonucleotide is extendedwith the DNA polymerase to produce a fourth DNA primer extension productcomplementary to the third DNA primer extension product. See FIG. 3,Step 16. Multiple copies of a second RNA product complementary to thethird DNA primer extension product, not including the promoter portion,are transcribed from the double-stranded promoter and re-enter theamplification cycle at Step 9 of FIG. 3. The base sequence of the secondRNA product is substantially identical to the base sequence of the tagsequence and the complement of the target sequence.

FIG. 5 illustrates an adaptation of an rTMA amplification reaction foramplifying a DNA target sequence, various aspects of which are disclosedin Becker et al., U.S. Pat. No. 7,713,697. The reaction of thisillustrative embodiment is initiated by treating a DNA target sequencein a nucleic acid sample with a tagged priming oligonucleotide and aterminating oligonucleotide. The tagged priming oligonucleotide includesa target hybridizing sequence hybridized to a 3′-end of the targetsequence and a tag sequence situated 5′ to the target hybridizingsequence. The target hybridizing sequence preferably hybridizes to asingle-stranded form of the target sequence, although it may hybridizeto a double-stranded form of the target sequence through strandinvasion, which can be facilitated by, for example, DNA breathing (e.g.,AT rich regions), low salt conditions, and/or the use of DMSO and/orosmolytes, such as betaine. The target sequence is preferably renderedsingle-stranded by heating the nucleic acid sample. The terminatingoligonucleotide hybridizes to a region of a target nucleic acidcontaining the target sequence in the vicinity of the 5′-end of thetarget sequence. The terminating oligonucleotide is used to end primerextension of a nascent nucleic acid that includes the tagged primingoligonucleotide. Thus, the target nucleic acid forms a stable complexwith the tagged priming oligonucleotide at the 3′-end of the targetsequence and the terminating oligonucleotide located adjacent to or nearthe 5′-end of the target sequence. See FIG. 5, Steps 1-3. Unhybridizedtagged priming oligonucleotide is made unavailable for hybridization tothe target sequence prior to initiating a primer extension reaction withthe tagged priming oligonucleotide, preferably by inactivating and/orremoving the unhybridized tagged priming oligonucleotide from thenucleic acid sample.

An extension reaction is then initiated from the 3′-end of the taggedpriming oligonucleotide with a DNA polymerase, e.g., reversetranscriptase, to produce a first DNA primer extension product thatincludes the tag sequence and a region complementary to the targetsequence. See FIG. 5, Steps 4 and 5.

The nucleic acid sample is further treated with a displaceroligonucleotide which hybridizes to the target nucleic acid upstreamfrom the tagged oligonucleotide such that a primer extension reactioncan be initiated therefrom, so that the first DNA primer extensionproduct is displaced when a 3′-end of the displacer oligonucleotide isextended by the DNA polymerase. See FIG. 5, Steps 6-8. The order of theillustrated steps is not meant to imply that the nucleic acid sample ofthis embodiment must be treated with the tagged priming oligonucleotidebefore it is treated with the displacer oligonucleotide to beoperational. In certain embodiments, it is preferable to have these twooligonucleotides hybridize to the target nucleic acid substantiallysimultaneously.

Next, the first DNA primer extension product is treated with a promoteroligonucleotide having a hybridizing sequence and a promoter for an RNApolymerase situated 5′ to the hybridizing sequence. The hybridizingsequence hybridizes to a region of the first DNA primer extensionproduct that is complementary to the 3′-end of the target sequence,thereby forming a promoter oligonucleotide:first DNA primer extensionproduct hybrid. In the illustrated reaction, the promoteroligonucleotide is modified to prevent the initiation of DNA synthesisby situating a blocking moiety at the 3′-end of the promoteroligonucleotide (e.g., nucleotide sequence having a 3′-to-5′orientation). See FIG. 5, Step 9. The 3′-end of the first DNA primerextension product is extended to add sequences complementary to thepromoter, resulting in the formation of a double-stranded promotersequence. See FIG. 5, Steps 10 and 11. Multiple copies of a first RNAproduct complementary to at least a portion of the first DNA primerextension product, not including the promoter, are transcribed using anRNA polymerase which recognizes the double-stranded promoter andinitiates transcription therefrom. See FIG. 5, Step 12 and 13. As aresult, the base sequence of the first RNA product is substantiallyidentical to the base sequence of the target sequence and the complementof the tag sequence.

The first RNA products are contacted with a priming oligonucleotidewhich hybridizes to the complement of the tag sequence to form a primingoligonucleotide:first RNA product hybrid, and the 3′-end of the primingoligonucleotide is extended with the DNA polymerase to produce a secondDNA primer extension product complementary to the first RNA product. SeeFIG. 5, Step 14-16. The second DNA primer extension product is separatedfrom the first RNA product using and enzyme that selectively degradesthe first RNA product (e.g., RNAse H activity). See FIG. 5, Step 17.

The second DNA primer extension product is treated with the promoteroligonucleotide to form a promoter oligonucleotide:second DNA primerextension product hybrid. See FIG. 5, Step 18. The promoteroligonucleotide:second primer extension product hybrid then re-entersthe amplification cycle at Step 10 of FIG. 5, where transcription isinitiated from the double-stranded promoter and the cycle continues.

FIG. 7 illustrates an adaptation of a polymerase chain reaction (PCR),various aspects of which are disclosed in, for example, Mullis et al.,U.S. Pat. Nos. 4,683,195 and 4,800,159; Mullis, U.S. Pat. No. 4,682,202;and Gelfand et al., U.S. Pat. No. 5,804,375. The reaction of thisillustrative embodiment is initiated by treating a denatured DNA targetsequence in a nucleic acid sample with a tagged priming oligonucleotide.The tagged priming oligonucleotide includes a target hybridizingsequence that hybridizes to a 3′-end of the target sequence and a tagsequence situated 5′ to the target hybridizing sequence. Thus, thetarget sequence forms a stable complex with the tagged primingoligonucleotide at the 3′-end of the target sequence prior to initiatinga primer extension reaction. See FIG. 7, Steps 1-3. Unhybridized taggedpriming oligonucleotide is made unavailable for hybridization to thetarget sequence prior to initiating a primer extension reaction with thetagged priming oligonucleotide, preferably by inactivating and/orremoving the unhybridized tagged priming oligonucleotide from thenucleic acid sample.

An extension reaction is then initiated from the 3′-end of the taggedpriming oligonucleotide with a DNA polymerase, e.g., Taq DNA polymerase,to produce a first DNA primer extension product that includes the tagsequence and a region complementary to the target sequence. See FIG. 7,Steps 4 and 5. Next, the double-stranded product resulting from thefirst primer extension reaction is denatured and the first DNA primerextension product is contacted with a first priming oligonucleotidewhich hybridizes to a region of the first DNA primer extension productthat is complementary to the 5′-end of the target sequence. See FIG. 7,Steps 6 and 7.

In a second primer extension reaction, the 3′-end of the first primingoligonucleotide is extended with the DNA polymerase to produce a secondDNA primer extension product that is complementary to a portion of thefirst primer extension product and includes the target sequence and thecomplement of the tag sequence. See FIG. 7, Steps 8 and 9. Thedouble-stranded product resulting from the second primer extensionreaction is denatured and the second DNA primer extension product iscontacted with a second priming oligonucleotide that hybridizes to thecomplement of the tag sequence. See FIG. 7, Steps 10 and 11.

The 3′-end of the second priming oligonucleotide is then extended in athird primer extension reaction with the DNA polymerase to produce athird DNA primer extension product that is complementary to the secondDNA primer extension product. FIG. 7, Steps 12 and 13. Thedouble-stranded product resulting from the third primer extensionreaction is denatured and the second and third DNA primer extensionproducts are available for participation in the repeated cycles of apolymerase chain reaction using as primers the first and second primingoligonucleotides. See FIG. 7, Steps 14-16.

FIG. 9 illustrates an adaptation of a reverse transcription polymerasechain reaction (RT-PCR), various aspects of which are disclosed in, forexample, Gelfand et al., U.S. Pat. Nos. 5,322,770 and 5,310,652. Thereaction of this illustrative embodiment is initiated by treating an RNAtarget sequence in a nucleic acid sample with a tagged primingoligonucleotide. The tagged priming oligonucleotide includes a targethybridizing sequence and a tag sequence situated 5′ to the targethybridizing sequence. Thus, the target sequence forms a stable complexwith the tagged priming oligonucleotide at the 3′-end of the targetsequence prior to initiating a primer extension reaction. See FIG. 9,Step 1. Unhybridized tagged priming oligonucleotide is made unavailablefor hybridization to the target sequence prior to initiating a primerextension reaction with the tagged priming oligonucleotide, preferablyby inactivating and/or removing the unhybridized tagged primingoligonucleotide from the nucleic acid sample.

An extension reaction is then initiated from the 3′-end of the taggedpriming oligonucleotide with a DNA polymerase, e.g., MasterAmp™ Tth DNAPolymerase, to produce a first DNA primer extension product thatincludes the tag sequence and a region complementary to the targetsequence. See FIG. 9, Steps 2 and 3. The first DNA primer extensionproduct is then separated from the target nucleic acid sequence to whichit is hybridized using an enzyme that selectively degrades that portionof a target nucleic acid containing the target sequence that iscomplementary to the first DNA primer extension product (e.g., RNAse Hactivity). See FIG. 9, Step 4.

Next, the first DNA primer extension product is treated with a firstpriming oligonucleotide which hybridizes to a region of the first DNAprimer extension product that is complementary to the 5′-end of thetarget sequence to form a first DNA primer extension product:firstpriming oligonucleotide hybrid. See FIG. 9, Step 5. A second primerextension reaction extends the 3′-end of the first primingoligonucleotide with the DNA polymerase to produce a DNA second primerextension product complementary to at least a portion of the firstprimer extension product and includes the target sequence and thecomplement of the tag sequence. See FIG. 9, Steps 6 and 7. The first andsecond DNA primer extension products are then separated from each otherby denaturation. See FIG. 9, Step 8. The first and second extensionproducts are then available to participate in the repeated cycles of apolymerase chain reaction using as primers the first primingoligonucleotide and a second priming oligonucleotide which hybridizes tothe complement of the tag sequence. See FIG. 9, Steps 9 and 10; FIG. 7,Steps 13-16.

In other illustrative embodiments of the present invention, aheterologous tag sequence which has not formed part of a tagged targetnucleic acid sequence is inactivated prior to exposing the tagged targetnucleic acid sequence to reagents and conditions sufficient fordetectable amplification of a target nucleic acid sequence. In apreferred aspect, the inactivated heterologous tag sequence is in theform of a tagged oligonucleotide which has not hybridized to the targetnucleic acid sequence. Tagged oligonucleotides are described above andinclude first and second regions, where the first region comprises atarget hybridizing sequence which hybridizes to a 3′-end of a targetnucleic acid sequence under a first set of conditions, and the secondregion comprises a tag sequence which is located 5′ to the first regionof the tagged oligonucleotide. The target hybridizing sequence has afree 3′ hydroxyl group that can be enzymatically extended in thepresence of a DNA polymerase in a template-dependent manner. The taggedoligonucleotide has an “active” confirmation which permits the targethybridizing sequence to hybridize to the target nucleic acid sequenceand an “inactive” confirmation which blocks the target hybridizingsequence from hybridizing to the target nucleic acid sequence. Theinactive confirmation is generally formed under less stringentconditions than the conditions for forming the active confirmation ofthe tagged oligonucleotide.

The inactive confirmation of the tagged oligonucleotide can be formed byhybridizing a tag closing sequence to the target hybridizing sequence ofthe tagged oligonucleotide. The tag closing sequence can constitute adiscrete molecule or it can be tethered to the tagged oligonucleotide bya linker which joins the 3′-end or 5′-end of the tag closing sequence tothe 5′-end of a region of the tagged oligonucleotide containing a tagsequence (“tagged priming oligonucleotide”) or a promoter sequencelocated 5′ to a tag sequence (“tagged promoter oligonucleotide”),thereby forming a self-hybridized, hairpin tag molecule comprising thetagged oligonucleotide. The linker does not include nucleotide basesthat can be copied by a polymerase and is preferably a non-nucleotidelinker comprised of non-nucleotide constituents. Suitable non-nucleotidelinkers for joining the tag closing sequence to the taggedoligonucleotide include abasic nucleotides and polyethylene glycol.Other suitable linkers include nucleotide analogs, such as LNAs and2′-O-Me. The association kinetics are best when the tag closing sequenceand the target hybridizing sequence of the tagged oligonucleotide arecontained in the same molecule.

Under selective conditions, the tag closing sequence can hybridize tothe target hybridizing sequence of the tagged oligonucleotide in anantiparallel orientation, as shown in FIGS. 2, 4, 6, 8, 10, 11, 12, 15and 16, or in a parallel orientation, as shown in FIGS. 13 and 14. Ifthe tag closing sequence is a discrete molecule, as illustrated in FIGS.11 and 12, or joined to the tagged oligonucleotide by a non-nucleotidelinker attached to its 5′-end, as illustrated in FIGS. 2, 4, 6, 8, 10,15 and 16, then the tag closing sequence is preferably modified toprevent primer extension by a DNA polymerase, such as by positioning ablocking moiety at its 3′-terminus. Suitable blocking moieties aredescribed herein. When hybridized in an antiparallel orientation, asillustrated in FIGS. 13 and 14, the 3′-terminal base of the tag closingsequence is preferably hybridized to the 3′-terminal base of the targethybridizing sequence of the tagged oligonucleotide. More preferably, thetag closing sequence is modified to prevent primer extension by a DNApolymerase.

The length and base content of the tag closing sequence are selected sothat hybridization of the tagged oligonucleotide to the target nucleicacid sequence is favored under a first set of conditions and, when thetagged oligonucleotide is not hybridized to the target nucleic acidsequence, so that the tag closing sequence can form a stable hybrid withthe target hybridizing sequence under a second, less stringent set ofconditions. The tag closing sequence should be selected so that it isnot readily displaced from the target hybridizing sequence under theamplification conditions to which it may be subjected. Typically, thetag closing sequence will hybridize to from 5 to 20 contiguous ornon-contiguous bases of the target hybridizing sequence. Suitable tagclosing sequences preferably range from 5 to 15 bases in length. To biasthe target hybridizing sequence toward the target nucleic acid under thefirst set of conditions, the tag closing sequence may include, forexample, one or more abasic nucleotides, base mismatches or members ofwobble base pairs. Tag closing sequences are preferably selected tospecifically hybridize to the target hybridizing sequence more stronglythan any non-specific interactions with other nucleic acids present inan amplification reaction.

Following inactivation, inactive tagged oligonucleotides are preferablyremoved from a sample to limit unintended interactions with targetnucleic acid sequences entering the sample from a potentiallycontaminating source. Removal can be accomplished by immobilizing targetnucleic acids in a sample on a solid support and then removing othercomponents of the sample, including inactivated tagged oligonucleotides.To ensure that inactive tagged oligonucleotides are removed, the numberof non-specific interactions between the solid support and nucleic acidspresent in the sample should be limited. Any known solid support may beused for sample processing, such as matrices and particles that are freein solution. Particularly preferred supports are magnetic spheres thatare monodisperse (i.e., uniform in size±5%), thereby providingconsistent results, which is particularly advantageous for use in anautomated procedure.

Particularly preferred amplification techniques for incorporating thetagged oligonucleotides of the present invention include isothermalamplification reactions, such as TMA and variations of TMA, likereal-time TMA, which incorporates one or more feature of the methodsdescribed by Becker et al., U.S. Pat. No. 7,374,885, and Becker et al.,U.S. Pat. No. 7,713,697. For example, certain preferred real-time TMAmethods include the use of blocking moieties, terminating moieties,and/or other modifying moieties that provide improved TMA processsensitivity and accuracy.

Promoter oligonucleotides may be modified to prevent the synthesis ofDNA therefrom. For example, a promoter oligonucleotide may comprise ablocking moiety attached at its 3′-terminus to prevent primer extensionin the presence of a polymerase. In one example, at least about 80% ofthe oligonucleotides present in the amplification reaction whichcomprise a promoter further comprise a 3′-blocking moiety. In anotherembodiment, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% ofthe oligonucleotides provided to the amplification reaction whichcomprise a promoter are further modified to comprise a 3′-blockingmoiety. In another embodiment, any oligonucleotide used in anamplification reaction of the present invention which comprises apromoter sequence further comprises a 3′-terminus blocking moiety.

Certain embodiments of the present invention relate to amplification ofa target nucleic acid comprising an RNA target sequence. In some cases,the target nucleic acid has indeterminate 3′- and 5′-ends relative tothe desired RNA target sequence. The target nucleic acid is treated witha priming oligonucleotide which has a base region sufficientlycomplementary to a 3′-end of the RNA target sequence to hybridizetherewith and, as discussed above, further comprises a heterologous tagsequence in the first primer extension reaction. Primingoligonucleotides are designed to hybridize to a suitable region of anydesired target sequence, according to primer design methods well knownto those of ordinary skill in the art. While the presence of the tagsequence in a priming oligonucleotide may alter the bindingcharacteristics of a target hybridizing region to a target nucleic acidsequence, the artisan skilled in the molecular arts can readily designpriming oligonucleotides which contain both target hybridizing regionsand tag sequences that can be used in accordance with the methodsdescribed herein. Suitable priming oligonucleotides are described inmore detail herein. Additionally, the 5′-end of a primingoligonucleotide (preferably not a tagged priming oligonucleotide) mayinclude one or modifications which improve the binding properties (e.g.,hybridization or base stacking) of the priming oligonucleotide to a DNAextension product or to an RNA amplification product, as discussed morefully infra, provided the modifications do not substantially interferewith the priming function of the priming oligonucleotide or cleavage ofan RNA amplification product to which the priming oligonucleotide ishybridized. The 3′-end of the priming oligonucleotide is extended by anappropriate DNA polymerase, e.g., an RNA-dependent DNA polymerase(“reverse transcriptase”) in an extension reaction using the RNA targetsequence or amplification product as a template to give a DNA primerextension product which is complementary to the RNA template oramplification product.

DNA primer extension products are separated (at least partially) from anRNA template using an enzyme which degrades the RNA template oramplification product. Suitable enzymes, i.e., “selective RNAses,” arethose which act on the RNA strand of an RNA:DNA complex, and includeenzymes which comprise an RNAse H activity. Some reverse transcriptasesinclude an RNAse H activity, including those derived from Moloney murineleukemia virus and avian myeloblastosis virus. According to preferredamplification embodiments, the selective RNAse may be provided as anRNAse H activity of a reverse transcriptase, or may be provided as aseparate enzyme, e.g., as an E. coli RNAse H or a T. thermophilus RNAseH. Other enzymes which selectively degrade RNA present in an RNA:DNAduplex may also be used.

When the target sequence is DNA, a DNA primer extension product can beseparated from the template by treating the target nucleic acid with adisplacer oligonucleotide. The displacer oligonucleotide has a primingfunction and is designed to hybridize to the target nucleic acidupstream from the priming oligonucleotide (referred to as the “forwardpriming oligonucleotide” in this embodiment). By “upstream” is meantthat a 3′-end of the displacer oligonucleotide hybridizes to the targetnucleic acid upstream from a 3′-end of the forward primingoligonucleotide. Thus, the displacer oligonucleotide and the forwardpriming oligonucleotide may hybridize to overlapping or distinct regionsof the target nucleic acid. In preferred embodiments, the 3′-terminus ofthe displacer oligonucleotide is adjacent to or spaced up to 5 to 35bases from the 5′-terminus of the forward priming oligonucleotiderelative to the target nucleic acid (i.e., the target nucleic acid hasup to 5 to 35, contiguous unbound nucleotides situated between the3′-terminal base of the displacer oligonucleotide and the 5′-terminalbase of the priming oligonucleotide when both oligonucleotides arehybridized to the target nucleic acid). The displacer oligonucleotide isgenerally from 10 to 50 nucleotides in length and may include one ormore modifications at the 5′-end which improve the binding properties(e.g., hybridization or base stacking) of the displacer oligonucleotideto the target nucleic acid, provided that the modifications do notsubstantially interfere with the priming function of the displaceroligonucleotide. The displacer oligonucleotide and the forward primingoligonucleotide are designed to hybridize to the target nucleic acidunder the same conditions. The target nucleic acid is preferably treatedwith the displacer oligonucleotide after the forward primingoligonucleotide has had sufficient time to hybridize to the targetnucleic acid. Alternatively, the target nucleic acid is treated withboth the displacer oligonucleotide and the forward primingoligonucleotide before exposing the mixture to a polymerase suitable forextending the 3′-ends of the displacer oligonucleotide and the forwardpriming oligonucleotide. In the presence of the DNA polymerase, the3′-end of the displacer oligonucleotide is extended in atemplate-dependent manner to form a second DNA primer extension productwhich displaces the first DNA primer extension product from the targetnucleic acid, thereby making it available for hybridization to apromoter oligonucleotide. In an alternative approach, conditions couldbe established whereby the promoter oligonucleotide gains access thefirst DNA primer extension product through stand invasion facilitatedby, for example, DNA breathing (e.g., AT rich regions), low saltconditions, and/or the use of DMSO and/or osmolytes, such as betaine.The promoter oligonucleotide of this embodiment is the same as thatdescribed above and, likewise, is modified to prevent the promoteroligonucleotide from functioning as a priming oligonucleotide for a DNApolymerase (e.g., the promoter oligonucleotide includes a blockingmoiety at its 3′-terminus).

In certain embodiments, the methods of the present invention furthercomprise treating the target nucleic acid as described above to limitthe length of the DNA primer extension product to a certain desiredlength. Such length limitation is typically carried out through use of a“binding molecule” which hybridizes to or otherwise binds to the RNAtarget nucleic acid adjacent to or near the 5′-end of the desired targetsequence. In certain embodiments, a binding molecule comprises a baseregion. The base region may be DNA, RNA, a DNA:RNA chimeric molecule, oran analog thereof. Binding molecules comprising a base region may bemodified in one or more ways, as described elsewhere herein. Suitablebinding molecules include, but are not limited to, a binding moleculecomprising a terminating oligonucleotide or a terminating protein thatbinds RNA and prevents primer extension past its binding region, or abinding molecule comprising a modifying molecule, for example, amodifying oligonucleotide such as a “digestion” oligonucleotide thatdirects hydrolysis of that portion of the RNA target hybridized to thedigestion oligonucleotide, or a sequence-specific nuclease that cuts theRNA target.

Illustrative terminating oligonucleotides of the present invention havea 5′-base region sufficiently complementary to the target nucleic acidat a region adjacent to, near to, or overlapping with the 5′-end of thetarget sequence, to hybridize therewith. In certain embodiments, aterminating oligonucleotide is synthesized to include one or moremodified nucleotides. For example, certain terminating oligonucleotidesof the present invention comprise one or more 2′-O-ME ribonucleotides,or are synthesized entirely of 2′-O-ME ribonucleotides. See, e.g.,Majlessi et al. (1998) Nucleic Acids Res., 26, 2224-2229. A terminatingoligonucleotide of the present invention typically also comprises ablocking moiety at its 3′-end to prevent the terminating oligonucleotidefrom functioning as a primer for a DNA polymerase. In some embodiments,the 5′-end of a terminating oligonucleotide of the present inventionoverlaps with and is complementary to at least about 2 nucleotides ofthe 5′-end of the target sequence. Typically, the 5′-end of aterminating oligonucleotide of the present invention overlaps with andis complementary to at least 3, 4, 5, 6, 7, or 8 nucleotides of the5′-end of the target sequence, but no more than about 10 nucleotides ofthe 5′-end of the target sequence. (As used herein, the term “end”refers to a 5′- or 3′-region of an oligonucleotide, nucleic acid ornucleic acid region which includes, respectively, the 5′- or 3′-terminalbase of the oligonucleotide, nucleic acid or nucleic acid region.)Suitable terminating oligonucleotides are described in more detailherein.

A single-stranded DNA primer extension product, or “first” DNA primerextension product, which has either a defined 3′-end or an indeterminate3′-end, is treated with a promoter oligonucleotide which comprises afirst region sufficiently complementary to a 3′-region of the DNA primerextension product to hybridize therewith, a second region comprising apromoter for an RNA polymerase, e.g., T7 polymerase, which is situated5′ to the first region, e.g., immediately 5′ to or spaced from the firstregion, and modified to prevent the promoter oligonucleotide fromfunctioning as a primer for a DNA polymerase (e.g., the promoteroligonucleotide includes a blocking moiety attached at its 3′-terminus).Upon identifying a desired hybridizing “first region,” suitable promoteroligonucleotides can be constructed by one of ordinary skill in the artusing only routine procedures. Those of ordinary skill in the art willreadily understand that a promoter region has certain nucleotides whichare required for recognition by a given RNA polymerase. In addition,certain nucleotide variations in a promoter sequence might improve thefunctioning of the promoter with a given enzyme, including the use ofinsertion sequences.

Insertion sequences may be positioned between the first and secondregions of promoter oligonucleotides and function to increaseamplification rates. (The tag sequence of a tagged promoteroligonucleotide may provide this beneficial effect.) The improvedamplification rates may be attributable to several factors. First,because an insertion sequence increases the distance between the 3′-endand the promoter sequence of a promoter oligonucleotide, it is lesslikely that a polymerase, e.g., reverse transcriptase, bound at the3′-end of the promoter oligonucleotide will interfere with binding ofthe RNA polymerase to the promoter sequence, thereby increasing the rateat which transcription can be initiated. Second, the insertion sequenceselected may itself improve the transcription rate by functioning as abetter template for transcription than the target sequence. Third, sincethe RNA polymerase will initiate transcription at the insertionsequence, the primer extension product synthesized by the primingoligonucleotide, using the RNA transcription product as a template, willcontain the complement of the insertion sequence toward the 3′-end ofthe primer extension product. By providing a larger target bindingregion, i.e., one which includes the complement of the insertionsequence, the promoter oligonucleotide may bind to the primer extensionproduct faster, thereby leading to the production of additional RNAtranscription products sooner. Insertion sequences are preferably 5 to20 nucleotides in length and should be designed to minimizeintramolecular folding and intermolecular binding with otheroligonucleotides present in the amplification reaction mixture. Programswhich aid in minimizing secondary structure are well known in the artand include Michael Zucker's mfold software for predicting RNA and DNAsecondary structure using nearest neighbor thermodynamic rules. Thelatest version of Michael Zucker's mfold software can be accessed on theWeb at www.bioinfo.rpi.edu/applications/mfold using a hypertext transferprotocol (http) in the URL. Useful insertion sequences may be identifiedusing in vitro selection methods well known in the art without engagingin anything more than routine experimentation.

Assaying promoter oligonucleotides with variations in the promotersequences is easily carried out by the skilled artisan using routinemethods. Furthermore, if it is desired to utilize a different RNApolymerase, the promoter sequence in the promoter oligonucleotide iseasily substituted by a different promoter. Substituting differentpromoter sequences is well within the understanding and capabilities ofthose of ordinary skill in the art. For real-time TMA, promoteroligonucleotides provided to the amplification reaction mixture aremodified to prevent efficient initiation of DNA synthesis from their3′-termini, and preferably comprise a blocking moiety attached at their3′-termini. Furthermore, terminating oligonucleotides and cappingoligonucleotides, and even probes used in certain embodiments of thepresent invention also optionally comprise a blocking moiety attached attheir 3′-termini.

Where a terminating oligonucleotide is used, the first region of thepromoter oligonucleotide is designed to hybridize with a desired 3′-endof the DNA primer extension product with substantial, but notnecessarily exact, precision. Subsequently, the second region of thepromoter oligonucleotide may act as a template, allowing the first DNAprimer extension product to be further extended to add a base regioncomplementary to the second region of the promoter oligonucleotide,i.e., the region comprising the promoter sequence, rendering thepromoter double-stranded. An RNA polymerase which recognizes thepromoter binds to the promoter sequence, and initiates transcription ofmultiple RNA copies complementary to the DNA primer extension product,which copies are substantially identical to the target sequence. By“substantially identical” it is meant that the multiple RNA copies mayhave additional nucleotides either 5′ or 3′ relative to the targetsequence, or may have fewer nucleotides either 5′ or 3′ relative to thetarget sequence, depending on, e.g., the boundaries of “the targetsequence,” the transcription initiation point, or whether the primingoligonucleotide comprises additional nucleotides 5′ of the primer region(e.g., a linked “cap” as described herein). Where a target sequence isDNA, the sequence of the RNA copies is described herein as being“substantially identical” to the target sequence. It is to beunderstood, however, that an RNA sequence which has uridine residues inplace of the thymidine residues of the DNA target sequence still has a“substantially identical” sequence. The RNA transcripts so produced mayautomatically recycle in the above system without further manipulation.Thus, this reaction is autocatalytic. In those embodiments where abinding molecule or other means for terminating a primer extensionreaction is not used, the first region of the promoter oligonucleotideis designed to hybridize with a selected region of the first DNA primerextension product which is expected to be 5′ to the 3′-terminus of thefirst DNA primer extension product, but since the 3′-terminus of thefirst DNA primer extension product is indeterminate, the region wherethe promoter oligonucleotide hybridizes probably will not be at theactual 3′-end of the first DNA primer extension product. According tothis embodiment, it is generally the case that at least the 3′-terminalbase of the first DNA primer extension product does not hybridize to thepromoter oligonucleotide. Thus, according to this embodiment the firstDNA primer extension product will likely not be further extended to forma double-stranded promoter.

The formation of a double-stranded promoter sequence through extensionof a template nucleic acid is not necessary to permit initiation oftranscription of RNA complementary to the first DNA primer extensionproduct. The resulting “first” RNA products are substantially identicalto the target sequence, having a 5′-end defined by the transcriptioninitiation point, and a 3′-end defined by the 5′-end of the first DNAprimer extension product. A sufficient number of first RNA products areproduced to automatically recycle in the system without furthermanipulation. The priming oligonucleotide hybridizes to the 3′-end ofthe first RNA products, and is extended by a DNA polymerase to form asecond DNA primer extension product. Unlike the first DNA primerextension product formed without the use of a terminatingoligonucleotide or other binding molecule, the second DNA primerextension product has a defined 3′-end which is complementary to the5′-ends of the first RNA products. The second DNA primer extensionproduct is separated (at least partially) from the RNA template using anenzyme which selectively degrades the RNA template. The single-strandedsecond DNA primer extension product is then treated with a promoteroligonucleotide as described above, and the second region of thepromoter oligonucleotide acts as a template, allowing the second DNAprimer extension product to be further extended to add a base regioncomplementary to the second region of the promoter oligonucleotide,i.e., the region comprising the promoter sequence, rendering thepromoter double-stranded. An RNA polymerase which recognizes thepromoter binds to the promoter sequence, and initiates transcription ofmultiple “second” RNA products complementary to the second DNA primerextension product, and substantially identical to the target sequence.The second RNA transcripts so produced automatically recycle in theabove system without further manipulation. Thus, this reaction isautocatalytic.

In any of the embodiments described above, once a desired region for thetarget sequence is identified, that region can be analyzed to determinewhere selective RNAse degradation will optimally cause cuts or removalof sections of RNA from the RNA:DNA duplex. Analyses can be conducted todetermine the effect of the RNAse degradation of the target sequence byRNAse H activity present in AMV reverse transcriptase or MMLV reversetranscriptase, by an exogenously added selective enzyme with an RNAseactivity, e.g., E. coli RNAse H, or selective enzymes with an RNAseactivity from other sources, and by combinations thereof. Following suchanalyses, the priming oligonucleotide can be selected for so that itwill hybridize to a section of RNA which is substantially nondegraded bythe selective RNAse present in the reaction mixture, because substantialdegradation at the binding site for the priming oligonucleotide couldinhibit initiation of DNA synthesis and prevent optimal extension of theprimer. In other words, a priming oligonucleotide is typically selectedto hybridize with a region of the RNA target nucleic acid or thecomplement of the DNA target nucleic acid, located so that when the RNAis subjected to selective RNAse degradation, there is no substantialdegradation which would prevent formation of the primer extensionproduct.

Conversely, the site for hybridization of the promoter oligonucleotidemay be chosen so that sufficient degradation of the RNA strand occurs topermit efficient hybridization of the promoter oligonucleotide to theDNA strand. Typically, only portions of RNA are removed from the RNA:DNAduplex through selective RNAse degradation and, thus, some parts of theRNA strand will remain in the duplex. Selective RNAse degradation on theRNA strand of an RNA:DNA hybrid results in the dissociation of smallpieces of RNA from the hybrid. Positions at which RNA is selectivelydegraded may be determined through standard hybridization analyses.Thus, a promoter oligonucleotide may be selected which will moreefficiently bind to the DNA after selective RNAse degradation, i.e.,will bind at areas where RNA fragments are selectively removed.

Promoters or promoter sequences suitable for incorporation in promoteroligonucleotides used in the methods of the present invention arenucleic acid sequences (either naturally occurring, producedsynthetically or a product of a restriction digest) that arespecifically recognized by an RNA polymerase that recognizes and bindsto that sequence and initiates the process of transcription, whereby RNAtranscripts are produced. Typical, known and useful promoters includethose which are recognized by certain bacteriophage polymerases, such asthose from bacteriophage T3, T7, and SP6, and a promoter from E. coli.The sequence may optionally include nucleotide bases extending beyondthe actual recognition site for the RNA polymerase which may impartadded stability or susceptibility to degradation processes or increasedtranscription efficiency. Promoter sequences for which there is a knownand available polymerase that is capable of recognizing the initiationsequence are particularly suitable to be employed.

Suitable DNA polymerases for use in accordance with the methods of theinvention include reverse transcriptases. Particularly suitable DNApolymerases include AMV reverse transcriptase and MMLV reversetranscriptase. Some of the reverse transcriptases suitable for use inthe methods of the present invention, such as AMV and MMLV reversetranscriptases, have an RNAse H activity. Indeed, according to certainembodiments of the present invention, the only selective RNAse activityin the amplification reaction is provided by the reversetranscriptase—no additional selective RNAse is added. However, in somesituations it may also be useful to add an exogenous selective RNAse,such as E. coli RNAse H. Although the addition of an exogenous selectiveRNAse is not required, under certain conditions, the RNAse H activitypresent in, e.g., AMV reverse transcriptase may be inhibited orinactivated by other components present in the reaction mixture. In suchsituations, addition of an exogenous selective RNAse may be desirable.For example, where relatively large amounts of heterologous DNA arepresent in the reaction mixture, the native RNAse H activity of the AMVreverse transcriptase may be somewhat inhibited and thus the number ofcopies of the target sequence produced accordingly reduced. Insituations where the target nucleic acid comprises only a small portionof the nucleic acid present (e.g., where the sample contains significantamounts of heterologous DNA and/or RNA), it is particularly useful toadd an exogenous selective RNAse. See, e.g., Kacian et al, U.S. Pat. No.5,399,491.

RNA amplification products produced by the methods described above mayserve as templates to produce additional amplification products relatedto the target sequence through the above-described mechanisms. Thesystem is autocatalytic and amplification by the methods of the presentinvention occurs without the need for repeatedly modifying or changingreaction conditions such as temperature, pH, ionic strength and thelike. These methods do not require an expensive thermal cyclingapparatus, nor do they require several additions of enzymes or otherreagents during the course of an amplification reaction.

As noted above, the methods of the present invention are useful inassays for detecting and/or quantitating specific nucleic acid targetsequences in clinical, water, environmental, industrial, beverage, food,seed stocks and other samples or to produce large numbers of RNAamplification products from a specific target sequence for a variety ofuses. For example, the present invention is useful to screen clinicalsamples (e.g., blood, urine, feces, saliva, semen, or spinal fluid),food, water, laboratory and/or industrial samples for the presence ofspecific nucleic acids, specific organisms (e.g., using species-specificoligonucleotides) and/or specific classes of organisms in applicationssuch as in sterility testing (e.g., using universal oligonucleotides).The present invention can be used to detect the presence of, forexample, viruses, bacteria, fungi, or parasites.

The amplification product can be detected by any conventional means. Forexample, amplification product can be detected by hybridization with adetectably labeled probe and measurement of the resulting hybrids.Design criteria in selecting probes for detecting particular targetsequences are well known in the art and are described in, for example,Hogan et al., “Methods for Making Oligonucleotide Probes for theDetection and/or Quantitation of Non-Viral Organisms,” U.S. Pat. No.6,150,517. Hogan teaches that probes should be designed to maximizehomology for the target sequence(s) and minimize homology for possiblenon-target sequences. To minimize stability with non-target sequences,Hogan instructs that guanine and cytosine rich regions should beavoided, that the probe should span as many destabilizing mismatches aspossible, and that the length of perfect complementarity to a non-targetsequence should be minimized. Contrariwise, stability of the probe withthe target sequence(s) should be maximized, adenine and thymine richregions should be avoided, probe:target hybrids are preferablyterminated with guanine and cytosine base pairs, extensiveself-complementarity is generally to be avoided, and the meltingtemperature of probe:target hybrids should be about 2-10° C. higher thanthe assay temperature.

In a particular embodiment, the amplification product can be assayed bythe Hybridization Protection Assay (“HPA”), which involves hybridizing achemiluminescent oligonucleotide probe to the target sequence, e.g., anacridinium ester-labeled (“AE”) probe, selectively hydrolyzing thechemiluminescent label present on unhybridized probe, and measuring thechemiluminescence produced from the remaining probe in a luminometer.See, e.g., Arnold et al., “Homogenous Protection Assay,” U.S. Pat. No.5,283,174 and NORMAN C. NELSON ET AL., NONISOTOPIC PROBING, BLOTTING,AND SEQUENCING, ch. 17 (Larry J. Kricka ed., 2d ed. 1995).

In further embodiments, the present invention provides quantitativeevaluation of the amplification process in real-time by methodsdescribed herein. Evaluation of an amplification process in “real-time”involves determining the amount of amplicon in the reaction mixtureeither continuously or periodically during the amplification reaction,and the determined values are used to calculate the amount of targetsequence initially present in the sample. There are a variety of methodsfor determining the amount of initial target sequence present in asample based on real-time amplification. These include those disclosedby Wittwer et al., “Method for Quantification of an Analyte,” U.S. Pat.No. 6,303,305, and Yokoyama et al., “Method for Assaying Nucleic Acid,”U.S. Pat. No. 6,541,205. Another method for determining the quantity oftarget sequence initially present in a sample, but which is not based ona real-time amplification, is disclosed by Ryder et al., “Method forDetermining Pre-Amplification Levels of a Nucleic Acid Target Sequencefrom Post-Amplification Levels of Product,” U.S. Pat. No. 5,710,029.Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of example, “molecular torches” are a type ofself-hybridizing probe which includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification product under strand displacement conditions. Under stranddisplacement conditions, hybridization of the two complementary regions(which may be fully or partially complementary) of the molecular torchis favored, except in the presence of the target sequence, which willbind to the single-stranded region present in the target binding domainand displace all or a portion of the target closing domain. The targetbinding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and a variety of typesof interacting label pairs are disclosed by Becker et al., “MolecularTorches,” U.S. Pat. No. 6,534,274.

Another example of a detection probe having self-complementarity is a“molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complement sequence, an affinity pair (or nucleic acidarms) holding the probe in a closed conformation in the absence of atarget sequence present in an amplification product, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complement sequence separates themembers of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed by Tyagi et al., “Detectably Labeled Dual ConfirmationOligonucleotide Probes, Assays and Kits,” U.S. Pat. No. 5,925,517, andTyagi et al., “Nucleic Acid Detection Probes Having Non-FRETFluorescence Quenching and Kits and Assays Including Such Probes,” U.S.Pat. No. 6,150,097.

Other self-hybridizing probes for use in the present invention are wellknown to those of ordinary skill in the art. By way of example, probebinding pairs having interacting labels, such as those disclosed byMorrison, “Competitive Homogenous Assay,” U.S. Pat. No. 5,928,862 andGelfand et al., U.S. Pat. No. 5,804,375 for PCR reactions, might beadapted for use in the present invention. Additional detection systemsinclude “molecular switches,” as disclosed by Arnold et al.,“Oligonucleotides Comprising a Molecular Switch,” U.S. Pat. Appln. Pub.No. US 2005-0042638 A1. And other probes, such as those comprisingintercalating dyes and/or fluorochromes, might be useful for detectionof amplification products in the present invention. See, e.g., Ishiguroet al., “Method of Detecting Specific Nucleic Acid Sequences,” U.S. Pat.No. 5,814,447.

In those methods of the present invention where the initial targetsequence and the RNA transcription product share the same sense, it maybe desirable to initiate amplification before adding probe for real-timedetection. Adding probe prior to initiating an amplification reactionmay slow the rate of amplification since probe which binds to theinitial target sequence has to be displaced or otherwise remove duringthe primer extension step to complete a primer extension product havingthe complement of the target sequence. The initiation of amplificationis judged by the addition of amplification enzymes (e.g., a reversetranscriptase and an RNA polymerase).

In addition to the methods described herein, the present invention isdrawn to kits comprising one or more of the reagents required forcarrying out the methods of the present invention. Kits comprisingvarious components used in carrying out the present invention may beconfigured for use in any procedure requiring amplification of nucleicacid target molecules, and such kits can be customized for variousdifferent end-users. Suitable kits may be prepared, for example, formicrobiological analysis, blood screening, disease diagnosis, watertesting, product release or sterility testing, environmental orindustrial analysis, food or beverage testing, or for general laboratoryuse. Kits of the present invention provide one or more of the componentsnecessary to carry out nucleic acid amplifications according to theinvention. Kits may include reagents suitable for amplifying nucleicacids from one particular target or may include reagents suitable foramplifying multiple targets. Kits of the present invention may furtherprovide reagents for real-time detection of one or more nucleic acidtargets in a single sample, for example, one or more self-hybridizingprobes as described above. Kits may comprise a carrier that may becompartmentalized to receive in close confinement one or more containerssuch as vials, test tubes, wells, and the like. Preferably at least oneof such containers contains one or more components or a mixture ofcomponents needed to perform the amplification methods of the presentinvention.

A kit according to one embodiment of the present invention can include,for example, in one or more containers, a tagged oligonucleotide, aloneor in combination with a tag closing oligonucleotide or joined to a tagclosing sequence, a binding molecule or other means for terminating aprimer extension reaction, and, optionally, an extender oligonucleotideand/or a capping oligonucleotide. If real-time detection is used, theone or more containers may include one or more reagents for real-timedetection of at least one nucleic acid target sequence in a singlesample, for example, one or more self-hybridizing probes as describedabove. Another container may contain an enzyme reagent, such as a heatstable DNA polymerase for performing a PCR or RT-PCR reaction, or amixture of a reverse transcriptase (either with or without RNAse Hactivity), an RNA polymerase, and optionally an additional selectiveRNAse enzyme for a transcription-based amplification reaction. Theseenzymes may be provided in concentrated form or at workingconcentration, usually in a form which promotes enzyme stability. Theenzyme reagent may also be provided in a lyophilized form. See Shen etal., “Stabilized Enzyme Compositions for Nucleic Acid Amplification,”U.S. Pat. No. 5,834,254. Another one or more containers may contain anamplification reagent in concentrated form, e.g., 10×, 50×, or 100×, orat working concentration. An amplification reagent will contain one ormore of the components necessary to run the amplification reaction,e.g., a buffer, MgCl₂, KCl, dNTPs, rNTPs, EDTA, stabilizing agents, etc.Certain of the components, e.g., MgCl₂ and rNTPs, may be providedseparately from the remaining components, allowing the end user totitrate these reagents to achieve more optimized amplificationreactions. Another one or more containers may include reagents fordetection of amplification products, including one or more labeledoligonucleotide probes. Probes may be labeled in a number of alternativeways, e.g., with radioactive isotopes, fluorescent labels,chemiluminescent labels, nuclear tags, bioluminescent labels,intercalating dyes, or enzyme labels. In some embodiments, a kit of thepresent invention will also include one or more containers containingone or more positive and negative control target nucleic acids which canbe utilized in amplification experiments in order to validate the testamplifications carried out by the end user. In some instances, one ormore of the reagents listed above may be combined with an internalcontrol. Of course, it is also possible to combine one or more of thesereagents in a single tube or other containers. Supports suitable for usewith the invention, e.g., test tubes, multi-tube units, multi-wellplates, etc., may also be supplied with kits of the invention. Finally akit of the present invention may include one or more instructionmanuals.

Kits of the invention may contain virtually any combination of thecomponents set out above or described elsewhere herein. As one skilledin the art would recognize, the components supplied with kits of theinvention will vary with the intended use for the kits, and the intendedend user. Thus, kits may be specifically designed to perform variousfunctions set out in this application and the components of such kitswill vary accordingly.

The present invention is further drawn to various oligonucleotidesincluding, for example, the target specific oligonucleotides exemplifiedbelow. It is to be understood that oligonucleotides of the presentinvention may be DNA, RNA, DNA:RNA chimerics and analogs thereof, and,in any case, the present invention includes RNA equivalents of DNAoligonucleotides and DNA equivalents of RNA oligonucleotides.

Detection probes of the present invention may include, for example, anacridinium ester label, or labeled, self-hybridizing regions flankingthe sequence which hybridizes to the target sequence. In variousembodiments, these labeled oligonucleotide probes optionally orpreferably are synthesized to include at least one modified nucleotide,e.g., a 2′-O-ME ribonucleotide; or these labeled oligonucleotide probesoptionally or preferably are synthesized entirely of modifiednucleotides, e.g., 2′-O-ME ribonucleotides.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods,compositions, reaction mixtures and kits described herein are readilyapparent from the description of the invention contained herein in viewof information known to the ordinarily skilled artisan, and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES

Examples are provided below illustrating certain aspects and embodimentsof the invention. The examples below are believed to accurately reflectthe details of experiments actually performed, however, it is possiblethat some minor discrepancies may exist between the work actuallyperformed and the experimental details set forth below which do notaffect the conclusions of these experiments or the ability of skilledartisans to practice them. Skilled artisans will appreciate that theseexamples are not intended to limit the invention to the specificembodiments described therein. Additionally, those skilled in the art,using the techniques, materials and methods described herein, couldeasily devise and optimize alternative amplification systems forcarrying out these and related methods while still being within thespirit and scope of the present invention.

Unless otherwise indicated, oligonucleotides and modifiedoligonucleotides in the following examples were synthesized usingstandard phosphoramidite chemistry, various methods of which are wellknown in the art. See e.g., Carruthers et al. (1987) Meth. Enzymol. 154,287. Unless otherwise stated herein, modified nucleotides were 2′-O-MEribonucleotides, which were used in the synthesis as theirphosphoramidite analogs.

Example 1 Selective Amplification of HCV Using Tagged Oligonucleotidesin a Real-Time TMA Reaction

The following series of experiments were conducted to evaluate whetherthe use of a tagged oligonucleotide to modify a target nucleic acidsequence in a nucleic acid sample of interest prior to atranscription-mediated amplification reaction would permit the selectiveamplification of target nucleic acid sequence contributed by the nucleicacid sample of interest, while not amplifying target nucleic acidsequence contributed by sources other than the nucleic acid sample ofinterest.

Reagents and protocol conditions used in the performed experiments, aswell as a discussion of the results and conclusions of the experiments,are set forth below.

I. Oligonucleotides

Unless otherwise indicated, oligonucleotides were synthesized using anExpedite™ 8909 DNA Synthesizer (PerSeptive Biosystems, Framingham,Mass.) using standard phosphoramidite chemistry. See, e.g., Carrutherset al. (1987) Meth. Enzymol. 154, 287. Sequences are from 5′-to-3′. Theblocking moiety, if present, is at the 3′-end.

1. Tagged Priming Oligonucleotide:

(SEQ ID NO: 1) GTTTGTATGTCTGTTGCTATTATGTCTACAGGCATTGAGCGGGTTGATCCAAGAAAGGAC; 12 pmol/rxn

2. Priming Oligonucleotide:

(SEQ ID NO: 2) GTTTGTATGTCTGTTGCTATTAT; 12 pmol/rxn

3. Promoter Oligonucleotide:

(SEQ ID NO: 3) ATTTAATACGACTCACTATAGGGAGACCACAACGGTTTCTAGCCATGGCGTTAGTATGAG; 12 pmol/rxn

Blocking Moiety: A 3′-to-3′ linkage prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01)

4. Terminating Oligonucleotide:

(SEQ ID NO: 4) AmUmGmGmCmUmAmGmAmCmGmCmUmUmUmCmUmGmCmGmUmGmAmAmGmAm; 0.8 pmol/rxn

Blocking Moiety: Same as promoter oligonucleotide

5. Extender Oligonucleotide:

(SEQ ID NO: 5) TGTCGTGCAGCCTCCAGGACCCCCCCTCCCG GGAGAGCCATA; 12 pmol/rxn

Blocking Moiety: Same as promoter oligonucleotide

6. First Capture Probe:

(SEQ ID NO: 6) GmGmGmCmAmCmUmCmGmCmAmAmGmCmAmmCmCmCmUmTTTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA; 3 pmol/rxn

7. Second Capture Probe:

(SEQ ID NO: 7) CmAmUmGmGmUmGmCmAmCmGmGmUmCmUmAmCmGmTTTAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA; 3 pmol/rxn

8. Detection Probe:

(SEQ ID NO: 8) CmGmUmUmCmCmGmCmAmGmAmCmCmAmCmUmAmUm(Linker)GmAmAmCmGm; 4 pmol/rxn

Probe Type: Molecular Torch

Linker: 9-O-Dimethoxytrityl-triethylene glycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen ResearchCorporation, Sterling, Va.; Cat. No. 10-1909-90)

5′ Label: 6-Carboxyfluorescein (FAM) (BioGenex, San Ramon, Calif.; Cat.No. BGX-3008-01)

3′ Label: 4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL) (PrimeSynthesis, Inc., Aston, Pa.)

II. Reagents and Other Protocol Information

1. Amplification Reagent. The “Amplification Reagent” or “AMP Reagent”comprised 11.6 mM Trizma® base buffer, 15 mM Trizma® hydrochloridebuffer, 25 mM MgCl₂, 23.3 mM KCl₂, 3.33% (v/v) glycerol, 0.05 mM zincacetate, 0.76 mM dATP, 0.76 mM dCTP, 0.76 mM dGTP, 0.76 mM dTTP, 0.02%(v/v) ProClin 300 Preservative (Supelco, Bellefonte, Pa.; Cat. No.48126), 6.0 mM ATP, 6.0 mM CTP, 6.0 mM GTP, and 6.0 mM UTP, pH 7.81 to8.0 at 22° C.

2. Enzyme Reagent. The “Enzyme Reagent” comprised 70 mMN-acetyl-L-cysteine, 10% (v/v) TRITON® X-102 detergent, 16 mM HEPES, 3mM EDTA, 0.05% (w/v) sodium azide, 20 mM Trizma® base buffer, 50 mMKCl₂, 20% (v/v) glycerol, 165.6 mM trehalose, pH 7, and containing 224RTU/μL Moloney murine leukemia virus (“MMLV”) reverse transcriptase and140 U/μL T7 RNA polymerase, where one unit (i.e., RTU or U) of activityis defined as the synthesis and release of 5.75 fmol cDNA in 15 minutesat 37° C. for MMLV reverse transcriptase, and the production of 5.0 fmolRNA transcript in 20 minutes at 37° C. for T7 RNA polymerase.

3. Wash Solution. The “Wash Solution” comprised 10 mM HEPES, 6.5 mMNaOH, 1 mM EDTA, 0.3% (v/v) ethyl alcohol, 0.02% (w/v) methyl paraben,0.01% (w/v) propyl paraben, 150 mM NaCl, and 0.1% (w/v) sodium dodecylsulfate, pH 7.5.

4. Transport Medium. The “Transport Medium” comprised 150 mM HEPES, 8%(w/v) lithium lauryl sulfate, and 100 mM ammonium sulfate, pH 7.5.

5. Target Capture Reagent. The “Target Capture Reagent” or “TCR”comprised the components listed below. Additional information about theformulation of this mixture is described below under Target CaptureReagent Procedure (IIIA). The concentrations listed represent the finalconcentrations of the components after having been combined with themagnetic particle solution. The magnetic particles were Sera-Mag™ MG-CMCarboxylate Modified (Seradyn, Inc., Indianapolis, Ind.; Cat. No.24152105-050250), 1 micron, super-paramagnetic particles covalentlybound 5′-amino modified oligo(dT)₁₄. The HEPES, lithium hydroxide,lithium chloride, EDTA, lithium lauryl sulfate and ammonium sulfatecomponents were introduced with the TCR diluent and Transport Medium.

-   -   First Capture Probe; 15.0 nM    -   Second Capture Probe; 15.0 nM    -   Tagged Priming Oligonucleotide; 60.0 nM    -   Terminating Oligonucleotide; 4.0 nM    -   HEPES, Free Acid, Dihydrate; 118.7 mM    -   Lithium Hydroxide, Monohydrate; 98.9 mM    -   Lithium Chloride, High Purity; 470.6 mM    -   EDTA, Free Acid; 25.0 mM    -   Lithium Lauryl Sulfate; 110.2 mM    -   Ammonium Sulfate; 37.5 mM    -   Seradyn Poly dT14 Magnetic Particles; 0.075 ug/uL

6. Transcript Buffer. The “Transcript Buffer” comprised 0.2% lithiumlauryl sulfate.

7. Transcript Used. HCV Transcript

8. Product Numbers of Certain Materials or Equipment Used.

KingFisher™ Plate (Thermo Labsystems, Franklin, Mass.; Cat. No.97002540)

MJ Research microtiter plate (Bio-Rad Laboratories, Inc., Hercules,Calif.; Cat. No. HSP-9665)

Solo HT Incubator (Thermo Labsystems, Franklin, Mass.; Cat. No. 5161580)

KingFisher™ Comb (Thermo Labsystems, Franklin, Mass.; Cat. No. 97002510)

Eppendorf® Thermomixer R (Eppendorf North America; Westbury, N.Y.; Cat.No. 022670107 or 022670158)

DNA Engine Opticon® 2 Real-Time PCR Detection System (Bio-RadLaboratories, Inc., Hercules, Calif.; Cat. No. CFB-3220)

9. Additional Protocol Information.

For the described experiments, 3.3 μL of target-containing transcriptbuffer was added to each 2.0 ml microtube in step B6 below. The taggedpriming oligonucleotide and the terminating oligonucleotide were inwater before being added to the 2.0 mL microtubes. Samples were vortexedfor about 5 seconds. Incubating for 10 minutes at 60° C. was found to begenerally sufficient to capture the transcript. The plates were kept atroom temperature for 5 minutes following the 10 minute incubation toallow the plates to cool before the target capture steps. This is alsowhere the plates were transferred from the Solo HT Incubator to theKingFisher System. The speed of the thermomixer was 1400 rpm.

III. Target Capture Protocol

A. Target Capture Reagent (TCR) Procedure.

Magnetic beads were slowly mixed at room temperature (RT) for 45 minutesand 150 μL magnetic beads were added to 5 mL TCR diluent (15 ugbeads/r×n when 50 μL used per sample). The solution was slowly mixed atroom temperature for 35 minutes, at which time capture probe was addedto 5 mL of the TCR diluent (to a final concentration of 0.12 pmol/μL(6-pmol/50 μL r×n).

B. Sample Preparation.

AMP Reagent was prepared containing the promoter oligonucleotide,extender oligonucleotide and priming oligonucleotide (volume=1,600 μL).The solution was vortexed and placed at 2-8° C. until needed. Detectionprobe was prepared in Enzyme Reagent and placed at 2-8° C. until needed.Target dilutions were prepared in 0.2% LLS. 50 μL TCR was transferredinto 200 μL microplate wells. Each target copy level, tagged primingoligonucleotide and terminating oligonucleotide were added to 1.2 mL 50%Transport Medium, 50% H₂O in 2.0 mL microtubes. Target samples werevortexted and 150 μL transferred into 200 μL microplate (Plate 1) wellcontaining 50 μL TCR (each well contained zero or 1 million copies HCVtranscript plus appropriate amounts of tagged priming and terminatingoligonucleotides).

C. Target Capture Protocol.

The 200 μL microplate (Plate 1) was incubated at 60° C. for 10 minutesusing Labsystems Solo HT Incubator (Plate 1), and the microplate wasthen placed at RT for 5 minutes (Plate 1). 200 μL microplates (Plates 2& 3) were prepared with 200 uL Wash Reagent. Amplification plate (Plate4-MJ research 96 well microtiter plate) was prepared with 30 μL AMPReagent per well. The 96 well comb was placed into Plate 1. All fourplates were loaded into the KingFisher 96 unit and the target captureprotocol was initiated, as follows.

Plate 1 was mixed for 5 minutes at very slow speed and beads werecollected for 12 counts and then released into Plate 2 for 10 secondsusing slow speed. Plate 1 was then mixed for 1 second using very slowspeed, beads collected for 12 counts, and the beads were released intoPlate 2 for 10 seconds using slow speed.

Plate 2 was mixed for 30 seconds at medium speed and beads werecollected for 12 counts and then released into Plate 3 for 10 secondsusing very slow speed. Plate 2 was then mixed for 1 second at very slowspeed and beads were collected for 12 counts and released into Plate 3for 10 seconds using very slow speed.

Plate 3 was mixed for 30 seconds at medium speed, beads were collectedfor 12 counts, and the beads were released into Plate 4 for 10 secondsusing medium speed. Plate 3 was then mixed for 1 second at very slowspeed, beads collected for 12 counts and released into plate 4 for 10seconds using medium speed.

The 96 well microtiter plate (Plate 4) was removed and transferred tothe bench, covered with a sealing card, and placed in the DNA EngineOpticon® 2 Real-Time PCR Detection System (Bio-Rad Laboratories;Hercules, Calif.) (“real-time instrument”).

D. Real Time TMA.

Real-time TMA was performed as follows. The plate was incubated for 5minutes at 42° C. and then removed and placed on a 42° C. thermomixer.Each reaction well received a 10 μL aliquot of the Enzyme Reagent. Themicrotiter plate was covered with an adhesive tape seal, shaken gentlyfor 30 seconds on the thermomixer, and then placed into the real-timeinstrument at 42° C., where real-time assay monitoring was commenced.TTime values, which served as indicators of the amount of ampliconsynthesized, were determined from the monitored fluorescence signals.See Light et al., U.S. Pat. Appln. Pub. No. US 2006-0276972, paragraphs506-549.

IV. Results and Conclusion

Experiments were performed according to the procedures described abovefor detecting an HCV transcript (8 replicates). The TCR in each testcontained the same tagged priming oligonucleotide. A target capture stepwas performed for binding HCV transcript and removing unhybridizedtagged priming oligonucleotide and terminating oligonucleotide. Afterthe target capture step, an AMP Reagent was contacted with the beads ofthe TCR, with the AMP Reagent containing a priming oligonucleotidespecific for the complement of the tag sequence. No tagged primingoligonucleotide was included in this step.

Eight replicates were run for each condition. The detection probe wasadded via the Enzyme Reagent at 4 pmol per reaction. The HCV AMP Reagentcontained 12 pmol promoter oligonucleotide, 12 pmol extenderoligonucleotide and 12 pmol priming oligonucleotide per reaction.

The first set of experiments compared the results of reactions in whichno copies of the HCV transcript were spiked into the TCR or AMP Reagentwith the results of reactions in which 1×10⁶ copies of the HCVtranscript were spiked into the TCR. FIG. 17 shows the raw curves forHCV amplifications in which no target was spiked into the AMP Reagent.There was no detectable amplification when the HCV transcript was notspiked into the TCR or AMP Reagent, while the average TTime forreactions containing 1×10⁶ copies of the HCV transcript in the TCR was6.3 minutes. The “TTime” values relate to time of emergence (time atwhich signal rises above background), and a summary of these values forthe experiments performed is set forth in Table 1 below.

A second set of experiments compared the results of reactions in which1×10⁶ copies of the HCV transcript were spiked into the AMP Reagent onlywith reactions in which 1×10⁶ copies of the HCV transcript were spikedinto the TCR only. FIG. 18 shows the raw curves for HCV amplificationsin which target was spiked into the AMP Reagent. There was no detectableamplification when the HCV transcript was spiked into the AMP Reagent,while the average TTime for reactions containing 1×10⁶ copies of the HCVtranscript in the TCR was 6.3 minutes (Table 1). The zero target in TCsamples did not amplify, even with 1 million copies HCV transcriptspiked into the AMP Reagent.

A third set of experiments compared the results of reactions in which1×10⁶ copies of the HCV transcript and the tagged primingoligonucleotide were provided in the AMP Reagent (no copies of the HCVtranscript in TCR) with the results of reactions in which 1×10⁶ copiesof the HCV transcript were provided in the TCR and the tagged primingoligonucleotide was provided in the AMP Reagent. FIG. 19 shows that theAvgTTime for 1 million copies HCV transcript present only in the targetcapture step with tagged priming and terminating oligonucleotides spikedinto the AMP Reagent was 7.2 minutes. The zero samples with target,terminating oligonucleotide and tagged priming oligonucleotide spikedinto the AMP Reagent also produced robust amplification with anAvgTTime=8.6 minutes (Table 1).

TABLE 1 TTime Summary (AvgTTimes & SDTTimes) Target Target Avg. T SDTSample ID Name Amt Total RN1 TN1 Time Time 1 million target in TC-x6.0HCV 1E6 8 7 8 6.3 0.11 1 million target in TC, tagged HCV 1E6 8 8 8 7.20.20 non-T7 primer & terminating oligonucleotide in amp-x6.0 1 milliontarget in TC-x6.0 HCV 1E6 8 8 7 6.3 0.05 Zero target in TC, 1 millionHCV 0.00 8 8 0 N/A N/A target in amp-x0.0 Zero target in TC, 1 millionHCV 0.00 8 8 8 8.6 0.21 target, tagged non-T7 primer & terminatingoligonucleotide in amp-x0.0 Zero target in TC-x0.0 HCV 0.00 8 8 0 N/AN/A

The results of these experiments demonstrate that only when the taggedpriming oligonucleotide was present in the AMP Reagent along with thepriming oligonucleotide did zero TCR samples amplify when 1 millioncopies of HCV transcript were spiked into the AMP Reagent. Thus, HCVtranscript entering the system through the AMP Reagent is not amplifiedunless the tagged priming oligonucleotide is also provided with the AMPReagent.

The preceding Example demonstrated how a tagged priming oligonucleotidethat hybridized to an HCV template could be used for selectivelydetecting HCV nucleic acids in a sample of interest without interferencefrom contaminating nucleic acid introduced subsequent to a targetcapture step. The following Example illustrates how a similar approachwas used for detecting bacterial nucleic acids in a sample of interestdespite the presence of contaminating templates in reagents used forperforming the amplification reaction. Advantageously, non-complexedtagged priming oligonucleotide was substantially absent from thereaction mixture at the time a complex comprising the tagged primingoligonucleotide and the template contacted the DNA polymerase used inthe amplification reaction.

Example 2 below describes two procedures for amplifying E. coli rRNAnucleic acids, where the procedures differed by the use of both a taggedpriming oligonucleotide and target capture. The first procedure employedan E. coli specific non-tagged priming oligonucleotide in combinationwith a terminating oligonucleotide, a promoter oligonucleotide and adetection probe. The second procedure employed a tagged primingoligonucleotide having a target-complementary sequence identical to thatcontained in the E. coli specific non-tagged priming oligonucleotide ofthe first procedure, a tag-specific priming oligonucleotide, as well asa terminating oligonucleotide, a promoter oligonucleotide and adetection probe. The tag-specific priming oligonucleotide, which had anucleotide sequence corresponding to a segment of HIV-1, hybridized tothe complement of the tag sequence contained in the tagged primingoligonucleotide, but did not hybridize to the E. coli rRNA templatenucleic acid or the complement thereof. In the case of the secondprocedure, the terminating oligonucleotide, the promoter oligonucleotideand the detection probe were identical to those used in the firstprocedure. As demonstrated below, amplification reactions that omittedthe tagged priming oligonucleotide failed to discriminate betweensamples containing 0 and 10⁶ copies of a synthetic E. coli rRNA target.Conversely, the approach that included use of a tagged primingoligonucleotide and target capture clearly distinguished samplescontaining 0 and 10³ copies of the synthetic E. coli rRNA target.

Example 2 Use of a Tagged Priming Oligonucleotide Allows DiscriminationBetween Sample-Derived Templates and Exogenous Templates

A. Amplification Using a Non-Tagged Priming Oligonucleotide withoutTarget Capture

In a first procedure, amplification reactions employing a synthetic E.coli rRNA template were performed using a non-tagged primingoligonucleotide that hybridized to the template, a promoteroligonucleotide, a terminating oligonucleotide and a molecular torchdetection probe. Reactions were primed using the synthetic templateadded directly into the reaction mixtures (i.e., without undergoingtarget capture purification) at 0 or 10⁶ copies/reaction. A moleculartorch detection probe was used to monitor amplicon production as afunction of time. In the nucleotide sequences presented below,2′-O-methyl ribose (2′-O-Me) modifications of the polynucleotidebackbone are indicated by lower case “m.” Blocking moieties at the 3′termini of the promoter oligonucleotide and terminating oligonucleotidecomprised a 3′-to-3′ linkage that was prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). Oligonucleotides, reagents and essential methods used inthe procedure were as follows.

I. Oligonucleotides

1. Non-Tagged Priming Oligonucleotide:

CmUmGmCmTGGCACGGAGTTAGCCGGTGCTTC (SEQ ID NO: 9)

2. Promoter Oligonucleotide:

(SEQ ID NO: 10) ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG - block

3. Terminating Oligonucleotide:

(SEQ ID NO: 11) GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm - block

4. Detection Probe:

(SEQ ID NO: 12) ¹CmUmGmCmGmGmGmUmAmAmCmGmUmCmAmAmUmGmAmGmCmAmAmAm²CGCAG³ ¹fluorescein ²C9 linker ³DABCYL

5. Synthetic E. coli rRNA template:

(SEQ ID NO: 13) AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA

II. Reagents and Other Protocol Information

Amplification and enzyme reagents were essentially as described underExample 1. Procedures using the non-tagged priming oligonucleotide thathybridized to the E. coli template did not employ target captureoligonucleotides or reagents, did not employ transport medium or washsolution, and did not employ an extender oligonucleotide.

A. Real-Time Amplification Protocol.

Sample solutions were prepared using primerless amplification reagent,non-tagged priming oligonucleotide, promoter oligonucleotide,terminating oligonucleotide, detection probe and synthetic templatenucleic acid. Each well of a 96-well microtiter plate received a 30 μLaliquot of the prepared sample solution. The microtiter plate wascovered with an adhesive tape seal, incubated first for 10 minutes at60° C. in the DNA ENGINE OPTICON® 2 (Bio-Rad Laboratories; Hercules,Calif.) temperature-controlled real-time instrument, and thentemperature-adjusted to 42° C. for 5 minutes. Thereafter, the plate wasremoved from the real-time instrument and placed onto a 42° C.thermomixer. Each reaction well received a 10 μL aliquot of the enzymereagent. The microtiter plate was covered with an adhesive tape seal,shaken gently for 30 seconds on the thermomixer, and then placed intothe real-time instrument at 42° C. where real-time assay monitoring wascommenced. TTime values, which served as indicators of the amount ofamplicon synthesized, were determined from the monitored fluorescencesignals.

III. Results and Conclusion

As indicated in FIG. 20, substantially identical results were observedin reactions that included either 0 or 10⁶ copies of the templatenucleic acid, and so the assay showed no discrimination between thesetwo conditions. More specifically, fluorescent signals indicatingformation of E. coli nucleic acid amplification products emerged frombackground levels at substantially similar times (i.e., TTime=31.74minutes at the 0 copy level, and 31.19 minutes at the 10⁶ copy level) inboth reactions. Thus, a real-time amplification profile characteristicof high levels of the nucleic acid template was obtained even in theabsence of added E. coli rRNA template. This was consistent with thepresence of contaminating bacterial nucleic acid templates in one ormore of the reagents used for carrying out the amplification reactionsfollowing the target capture procedure.

B. Amplification Using a Tagged Priming Oligonucleotide and TargetCapture

In a second procedure, a tagged priming oligonucleotide and targetcapture step were employed for performing amplification reactions usingtest samples containing either 0, 10³ or 10⁵ copies of the synthetic E.coli transcript. Oligonucleotides used in the procedure are indicatedbelow. The molecular torch detection probe was added as a component ofthe enzyme reagent. Following target capture, tagged primingoligonucleotide that was not hybridized to template nucleic acid wasremoved from the system by standard target capture and wash steps. Thecomplex that included the rRNA template and the tagged primingoligonucleotide remained captured on super-paramagnetic particles.Amplification reactions were carried out using reagents essentially asdescribed above, except for substitution of a non-specific targetcapture probe for the sequence-specific capture probes employed inExample 1. Amplification reactions were carried out in replicates of sixand monitored using a molecular torch detection probe essentially asdescribed in Example 1, except that an extender oligonucleotide wasomitted. As above, 2′-O-methyl ribose (2′-O-Me) modifications of thepolynucleotide backbone in the sequences presented below are indicatedby lower case “m.” Blocking moieties at the 3′ termini of the promoteroligonucleotide and terminating oligonucleotide comprised a 3′-to-3′linkage that was prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). Oligonucleotides, reagents and essential methods used inthe procedure were as follows.

I. Oligonucleotides:

1. Tagged Priming Oligonucleotide:

(SEQ ID NO: 14) GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTAGCCGGTGCTTC

2. Tag-Specific Priming Oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 15)

3. Promoter Oligonucleotide:

(SEQ ID NO: 10) ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG - block

4. Terminating Oligonucleotide:

(SEQ ID NO: 11) GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm - block

5. Non-Specific Capture Probe:

(SEQ ID NO: 16) KmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

6. Detection Probe:

(SEQ ID NO: 12) ¹CmUmGmCmGmGmGmUmAmAmCmGmUmCmAmAmUmGmAmGmCmAmAmAm²CGCAG³ ¹fluorescein ²C9 linker ³DABCYL

7. Synthetic E. coli rRNA template

(See above)

II. Reagents and Other Protocol Information

Reagents and experimental protocols were essentially as described underExample 1, with the substitution of a non-specific target captureoligonucleotide for the first and second capture oligonucleotides, thesubstitution of the above-presented E. coli-specific oligonucleotidesfor HCV-specific oligonucleotides, and the omission of an extenderoligonucleotide.

III. Non-Specific Target Capture Protocol

A. Target Capture Reagent (TCR) Preparation.

A stock suspension of magnetic beads was mixed at room temperature for30 minutes. An aliquot of about 150 μL of the magnetic bead suspensionwas added to 5 mL of TCR diluent (15 μg beads/reaction when using 50μL/sample), and then slowly mixed at room temperature for 30 minutes.Next, the non-specific capture oligonucleotide was added to 5 mL of theTCR mixture to yield a final concentration of 0.12 pmol/μL. The preparedTCR was mixed gently at room temperature until needed.

B. Sample Preparation.

Amplification solution was prepared using primerless amplificationreagent, promoter oligonucleotide and tag-specific primingoligonucleotide. The prepared amplification solution was mixed byvortexing and then maintained at 2-8° C. until needed. Enzyme reagentcontaining the molecular torch detection probe was next prepared andmaintained at 2-8° C. until needed. Dilutions of the template rRNA wereprepared in 0.2% LLS (lithium lauryl sulfate). Aliquots (50 μL) of themagnetic bead target capture solution were transferred into the wells ofa microtiter plate for a KINGFISHER 96 (Thermo Fisher Scientific, Inc.;Waltham, Mass.) magnetic particle processor. Samples of dilutedtemplate, tagged priming oligonucleotide and terminating oligonucleotidewere then added to 1.5 mL of 50% transport medium diluted with water.The target-containing sample mixture was vortexed, and 150 μL aliquotstransferred into the microtiter plate (Plate 1) wells containing 50 μLtarget capture solution (each well contained 0, 10³ or 10⁵ copies of theE. coli transcript and the appropriate amount of tagged primingoligonucleotide and terminating oligonucleotide).

C. Target Capture Protocol.

First there was prepared a microtiter plate containing 200 μL of washreagent (Plate 2). Another microtiter plate (Plate 3) for conductingamplification reactions was prepared, with each well to be used for areaction containing 30 μL of amplification reagent. All three plates(Plates 1-3) were loaded into the magnetic particle processor unit.Magnetic beads harboring nucleic acid complexes were isolated from Plate1, washed in Plate 2, and then transferred into Plate 3 using standardprocedures familiar to those having an ordinary level of skill in theart. Plate 3 was removed from the magnetic particle processor unit,covered with an adhesive tape seal, and then placed into thetemperature-controlled real-time instrument.

D. Real-Time Amplification Protocol.

Plate 3 was incubated at 42° C. for 5 minutes in the real-timeinstrument. The microtiter plate was removed from the real-timeinstrument and placed onto a 42° C. thermomixer. Each reaction wellreceived a 10 μL aliquot of enzyme reagent containing detection probe,and was then covered with an adhesive tape seal. The plate was shakengently for 60 seconds on the thermomixer, and then placed back into thereal-time instrument at 42° C. where real time assay monitoring wascommenced. TTime values, which served as indicators of the amount ofamplicon synthesized, were determined from the monitored fluorescencesignals.

IV. Results and Conclusion

FIG. 21 graphically illustrates the benefits of the disclosed approachto nucleic acid amplification. Procedures that employed a tagged primingoligonucleotide complementary to a target of interest, a target capturestep, and a tag-specific priming oligonucleotide that was notcomplementary to the target of interest (i.e., the E. coli rRNA) yieldeddramatically reduced background amplification levels, and so easilypermitted discrimination between 0 and 10³ copies of the bacterialtemplate nucleic acid. More specifically, the average TTime valuesdetermined for reactions carried out using 10⁵ copies, 10³ copies, and 0copies of the E. coli template were 24.7 minutes, 30.6 minutes and 37.5minutes, respectively. Taken together with the results presented in FIG.4, these findings were consistent with the presence of bacteria-derivednucleic acids in common reagents used for conducting in vitro nucleicacid amplification reactions. Despite this fact, the procedure employinga tagged priming oligonucleotide was useful for detecting E. colinucleic acids contained in a test sample without interference fromexogenous template nucleic acids contributed by the amplificationreagents. For example, a qualitative assay for detecting E. coli nucleicacids at a level of 10³ copies or greater in a test sample could dependon achieving a threshold fluorescence signal or TTime value after apredetermined reaction time (e.g., 35 minutes).

The following Example presents comparative results showing how twodifferent detection probes influenced the profiles of real-timeamplification run curves. The results further showed how the taggedpriming oligonucleotide approach could be used for discriminating 0 and10³ copies of the synthetic E. coli template nucleic acid—a levelapproximating the number of copies of 16S rRNA present in a singlebacterium.

Example 3 describes detection of E. coli rRNA templates in real-timeamplification reactions using three different detection probes.

Example 3 Alternative Torch Designs can Improve Assay Results

Amplification reactions were conducted and monitored in a real-timeformat using one of three different detection probes. The synthetictemplate nucleic acid, non-specific capture oligonucleotide, taggedpriming oligonucleotide, termination oligonucleotide, promoteroligonucleotide and tag-specific priming oligonucleotide used forperforming the reactions were identical to those used in the secondprocedure of the preceding Example. The E. coli target hybridizingportion of the tagged priming oligonucleotide corresponded to nucleotidepositions 24-57 of SEQ ID NO:14 (i.e., the target hybridizing sequencecorresponding to SEQ ID NO:19). The E. coli target hybridizing portionof the promoter oligonucleotide corresponded to nucleotide positions27-47 of SEQ ID NO:10 (i.e., the target hybridizing sequencecorresponding to SEQ ID NO:20). Four replicates were run for eachcondition. As before, detection probe was added with the enzyme reagent.Reagents and protocols for non-specific target capture, samplepreparation, and real-time amplification also were essentially asdescribed in the second procedure of the preceding Example. Notably,reactions were conducted using 0, 10³ or 10⁵ copies of the synthetic E.coli template. As above, 2′-O-methyl ribose (OMe) modifications of thepolynucleotide backbone in the sequences presented below are indicatedby lower case “m.” Blocking moieties at the 3′ termini of the promoteroligonucleotide and terminating oligonucleotide comprised a 3′-to-3′linkage that was prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01). Oligonucleotides, reagents and essential methods used inthe procedure were as follows.

I. Oligonucleotides:

1. Tagged Priming Oligonucleotide:

(SEQ ID NO: 14) GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTAGCCGGTGCTTC

2. Tag-Specific Priming Oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 15)

3. Promoter Oligonucleotide:

(SEQ ID NO: 10) ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG - block

4. Terminating Oligonucleotide:

(SEQ ID NO: 11) GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm - block

5. Non-Specific Capture Probe:

(SEQ ID NO: 16) KmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

6. Detection Probe:

(SEQ ID NO: 17) ¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCm²GmCmUmCmGm³ (SEQ ID NO: 18) ¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCmUmUmUmAmCmUmCm²GmCmUmCmGm³ ¹fluorescein ²C9 linker ³DABCYL

7. Synthetic E. coli rRNA template

(See above)

II. Reagents and Other Protocol Information

Reagents and experimental protocols were essentially as described underExample 2, with a slight change to the conditions used for targetcapture.

III. Non-Specific Target Capture Protocol:

A. Target Capture Reagent (TCR) Preparation.

A stock suspension of magnetic beads was mixed at room temperature for25 minutes. A 150 μL aliquot of the magnetic bead suspension was addedto 5 mL of TCR diluent (15 μg beads/reaction when using50.micro.L/sample), and then slowly mixed at room temperature for 25minutes. Next, the non-specific capture oligonucleotide was added to 5mL of the TCR mixture to yield a final concentration of 0.12 pmol/μL.The prepared TCR was mixed gently at room temperature until needed.

B. Sample Preparation.

Amplification solutions were prepared using primerless AMP Reagent,promoter oligonucleotide and tag-specific priming oligonucleotide. Theprepared amplification solutions were mixed by vortexing and thenmaintained at 2-8° C. until needed. Enzyme Reagents containing themolecular torch detection probes were next prepared and maintained at2-8° C. until needed. Dilutions of the template rRNA were prepared in0.2% LLS, as described above. Aliquots (50 μL) of the magnetic beadtarget capture solution were transferred into the wells of a microtiterplate for a KINGFISHER 96 (Thermo Fisher Scientific, Inc.; Waltham,Mass.) magnetic particle processor. Samples of diluted template, taggedpriming oligonucleotide and terminating oligonucleotide were then addedto 1.5 mL of 50% Transport Medium diluted with water. Thetarget-containing sample mixture was vortexed, and 150 μL aliquotstransferred into the microtiter plate (Plate 1) wells containing 50 μLtarget capture solution (each well contained 0, 10³ or 10⁵ copies of theE. coli transcript and the appropriate amount of tagged primingoligonucleotide and terminating oligonucleotide).

C. Target Capture Protocol.

The microtiter plate (Plate 1) was incubated at 60° C. for 15 minutesusing a SOLO HT incubator (Thermo Labsystems; Franklin, Mass.). Themicrotiter plate was then placed on the bench at room temperature andallowed to equilibrate for 5 minutes (Plate 1). Next, there was prepareda second microtiter plate containing 200 μL of Wash Reagent (Plate 2). Athird microtiter plate (Plate 3) for conducting amplification reactionswas prepared, with each well to be used for a reaction containing 30 μLof amplification reagent. All three plates were loaded into the magneticparticle processor unit. Magnetic beads harboring nucleic acid complexeswere isolated from Plate 1, washed in Plate 2, and then transferred intoPlate 3 using standard procedures familiar to those having an ordinarylevel of skill in the art. Plate 3 was removed from the magneticparticle processor unit, covered with an adhesive tape seal, and thenplaced into the temperature-controlled real-time instrument.

D. Real-Time Amplification Protocol.

Plate 3 was incubated in the real-time instrument at 42° C. for 5minutes. The microtiter plate was removed from the real-time instrumentand placed onto the 42° C. thermomixer. Each reaction well received a 10μL aliquot of Enzyme Reagent containing detection probe, and was thencovered with an adhesive tape seal. The plate was shaken gently for 60seconds on the thermomixer, and then placed back into the real-timeinstrument at 42° C. where real-time assay monitoring was commenced.TTime values, which served as indicators of the amount of ampliconsynthesized, were determined from the monitored fluorescence signals.

IV. Results and Conclusion

The results presented in Table 2 summarize the average TTime values(column 3), and the standard deviations of the average TTime values(column 4) for reactions conducted using the different detection probes.The tabular summary confirmed that all of the tested detection probesyielded very good results in the real-time assays. Each probeadvantageously gave a very low signal at the 0 copy level of inputtarget. More specifically, amplicon detected in reactions carried outusing 0 copies of input synthetic template was essentially undetectablewhen the reactions included the detection probes of SEQ ID NO:17 and SEQID NO:18. Thus, reactions that included one of the detection probesidentified by SEQ ID NO:17 and SEQ ID NO:18 gave outstanding resultsthat easily permitted detection of template nucleic acids correspondingroughly to the amount contained in a single bacterium.

TABLE 2 Use of Alternative Detection Probes for Improved AssayDiscrimination Template Amount AvgTTime SDTTime (copies) Detection Probe(minutes) (minutes)  0 SEQ ID NO: 17 N/A N/A 10³ 38.2 2.81 10⁵ 26.4 0.32 0 SEQ ID NO: 18 N/A N/A 10³ 35.9 2.33 10⁵ 28.8 0.45

Taken in view of the results presented Examples 2 and 3, each of SEQ IDNos:12, and 17-18 represent preferred molecular torches for detecting E.coli using the methods described herein. Highly preferred probes usefulfor detecting E. coli nucleic acids will have target-complementarysequences corresponding to nucleotide positions 2-24 contained withinthe probe of SEQ ID NO:12 (i.e., the target hybridizing sequencecorresponding to SEQ ID NO:21), or nucleotide positions 2-17 containedwithin the probe of SEQ ID NO:17 (i.e., the target hybridizing sequencecorresponding to SEQ ID NO:22), or nucleotide positions 2-24 containedwithin the probe of SEQ ID NO:18 (i.e., the target hybridizing sequencecorresponding to SEQ ID NO:23). Generally speaking, probes useful fordetecting E. coli nucleic acids will have target hybridizing sequencesof at least 16 contiguous nucleotides contained within the sequence ofTGCGGGTAACGTCAATGAGCAAAGGTATTAACTTTACTC (SEQ ID NO:24). Overallpreferred lengths of desirable probes will be up to 39 nucleotides, morepreferably up to 29 nucleotides, more preferably up to 23 nucleotides,or still more preferably up to 16 nucleotides. Of course, useful probesmay include RNA and DNA equivalent bases, and include the complements ofthe foregoing described probes.

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

The invention claimed is:
 1. A method for the selective amplification ofat least one target nucleic acid sequence from a nucleic acid sample,said method comprising the steps of: (a) treating a nucleic acid samplecomprising a target nucleic acid sequence with a tagged oligonucleotidecomprising first and second regions, said first region comprising atarget hybridizing sequence which hybridizes to a 3′-end of said targetnucleic acid sequence and said second region comprising a tag sequencesituated 5′ to said target hybridizing sequence, wherein said secondregion does not stably hybridize to a target nucleic acid containingsaid target nucleic acid sequence, and wherein said target nucleic acidsequence is contained in each of a plurality of ribosomal nucleic acidsfrom multiple species of microorganisms; (b) prior to initiating aprimer extension reaction, reducing in said nucleic acid sample theeffective concentration of unhybridized tagged oligonucleotide having anactive form in which a target hybridizing sequence of said unhybridizedtagged oligonucleotide is available for hybridization to said targetnucleic acid sequence; and (c) after step (b), initiating an extensionreaction from the 3′-end of the tagged oligonucleotide with a DNApolymerase to produce a primer extension product comprising a regioncomplementary to the target nucleic acid sequence; (d) separating theprimer extension product from the target nucleic acid; and (e) producingamplification products in a nucleic acid amplification reaction usingfirst and second oligonucleotides, wherein said first oligonucleotidecomprises a hybridizing sequence which hybridizes to a 3′-end of thecomplement of said target nucleic acid sequence and said secondoligonucleotide comprises a hybridizing sequence which hybridizes to thecomplement of said tag sequence, wherein said second oligonucleotidedoes stably hybridize to said target nucleic acid, and wherein each ofsaid amplification products comprises a base sequence which issubstantially identical or complementary to the base sequence of saidtarget nucleic acid sequence and further comprises a base sequence whichis substantially identical or complementary to all or a portion of saidtag sequence.
 2. The method of claim 1, wherein step (b) comprisesremoving unhybridized tagged oligonucleotide from said nucleic acidsample.
 3. The method of claim 2, wherein said target nucleic acidsequence is immobilized on a solid support during step (b).
 4. Themethod of claim 1, wherein step (b) comprises inactivating unhybridizedtagged oligonucleotide so that said unhybridized tagged oligonucleotidedoes not stably hybridize to said target nucleic acid sequence duringstep (c).
 5. The method of claim 4 further comprising removingunhybridized tagged oligonucleotide from said nucleic acid sample duringstep (b).
 6. The method of claim 4, wherein said tagged oligonucleotidehas an active form during step (a) which permits said target hybridizingsequence to hybridize to said target nucleic acid sequence, and whereinunhybridized tagged oligonucleotide is converted to an inactive form instep (b) which blocks or prevents said tagged oligonucleotide fromhybridizing to said target nucleic acid sequence during step (c).
 7. Themethod of claim 6, wherein the conditions of steps (b)-(e) are lessstringent than the conditions of step (a).
 8. The method of claim 7,wherein the temperature of said nucleic acid sample is lowered betweensteps (a) and (b).
 9. The method of claim 6, wherein unhybridized taggedoligonucleotide from step (a) is converted from a single-stranded formto a duplexed form in step (b).
 10. The method of claim 9, wherein theduplexed form is hairpin tag molecule comprising a tag closing sequencejoined to a 5′-end of said tagged oligonucleotide, wherein said tagclosing sequence hybridizes to said target hybridizing sequence underthe conditions of step (b), thereby blocking hybridization ofunhybridized tagged oligonucleotide from step (a) to said target nucleicacid sequence in steps (b)-(e).
 11. The method of claim 10, wherein saidtag closing sequence is joined to said tagged oligonucleotide by anon-nucleotide linker.
 12. The method of claim 10, wherein a 3′-end ofsaid tag closing sequence is joined to a 5′-end of said taggedoligonucleotide.
 13. The method of claim 4, wherein said targethybridizing sequence is hybridized to a tag closing oligonucleotide instep (b), said tagged oligonucleotide and said tag closingoligonucleotide being distinct molecules.
 14. The method of claim 13,wherein said tag closing oligonucleotide is modified to prevent theinitiation of DNA synthesis therefrom.
 15. The method of claim 14,wherein a 3′-terminal base of said target hybridizing sequence ishybridized to a 5′-terminal base of said tag closing oligonucleotide.16. The method of claim 14, wherein said tagged oligonucleotide and saidtag closing oligonucleotide are both present in said nucleic acid sampleduring step (a), and wherein said target hybridizing sequence favorshybridization to said target nucleic acid sequence over said tag closingoligonucleotide in step (a).
 17. The method of claim 1, wherein theconditions of said nucleic acid amplification reaction are isothermal.18. The method of claim 17, wherein said nucleic acid amplificationreaction is a transcription-based amplification reaction.
 19. A kit foruse in the selective amplification of at least one target nucleic acidsequence from a nucleic acid sample, said kit comprising: (a) a taggedoligonucleotide comprising: (i) a first region comprising a targethybridizing sequence which hybridizes to a 3′-end of a target nucleicacid sequence under a first set of conditions so that said first regioncan be extended in a template-dependent manner in the presence of a DNApolymerase; and (ii) a second region comprising a tag sequence situated5′ to said first region, wherein said second region does not stablyhybridize to a target nucleic acid containing said target nucleic acidsequence under said first set of conditions; wherein said target nucleicacid sequence is contained in each of a plurality of ribosomal nucleicacids from multiple species of microorganisms; (b) a tag closingsequence which hybridizes to said target hybridizing sequence under asecond set of conditions, thereby blocking hybridization of said taggedoligonucleotide to said target nucleic acid sequence, wherein said tagclosing sequence does not stably hybridize to said target hybridizingsequence under said first set of conditions; and (c) a first primingoligonucleotide which hybridizes to the complement of said tag sequenceunder said second set of conditions so that said first primingoligonucleotide can be extended in a template-dependent manner in thepresence of a DNA polymerase.
 20. A pre-amplification reaction mixturefor selective amplification of one or more target nucleic acidsequences, said reaction mixture comprising: a tagged oligonucleotidecomprising first and second regions, said first region comprising atarget hybridizing sequence hybridized to a target region contained at a3′-end of a target nucleic acid sequence present in said reactionmixture and said second region comprising a tag sequence situated 5′ tosaid target hybridizing sequence, wherein said target nucleic acidsequence is contained in each of a plurality of ribosomal nucleic acidsfrom multiple species of microorganisms; a first oligonucleotidecomprising a hybridizing sequence which hybridizes to a 3′-end of thecomplement of said target nucleic acid sequence; and a secondoligonucleotide comprising a hybridizing sequence which hybridizes tothe complement of said tag sequence, wherein said reaction mixture issubstantially free of an active form of said tagged oligonucleotidewhich is not hybridized to said target region contained in said targetnucleic acid sequence present in said reaction mixture, wherein saidactive form of said tagged oligonucleotide has an available targethybridizing sequence for hybridization to said target region present ina non-target nucleic acid added to said reaction mixture, and whereinsaid reaction mixture does not comprise a nucleic acid polymerasecapable of extending any of said oligonucleotides in atemplate-dependent manner.